Methods and systems for processing polynucleotides

ABSTRACT

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/720,085, filed on Sep. 29, 2017, which claims priority to U.S.Provisional Patent Application No. 62/438,341, filed on Dec. 22, 2016,each of which is entirely incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 22, 2018, isnamed 43487742302SL.txt and is 22,715 bytes in size.

BACKGROUND

Significant advances in analyzing and characterizing biological andbiochemical materials and systems have led to unprecedented advances inunderstanding the mechanisms of life, health, disease and treatment.Among these advances, technologies that target and characterize thegenomic make up of biological systems have yielded some of the mostgroundbreaking results, including advances in the use and exploitationof genetic amplification technologies, and nucleic acid sequencingtechnologies.

Nucleic acid sequencing can be used to obtain information in a widevariety of biomedical contexts, including diagnostics, prognostics,biotechnology, and forensic biology. Sequencing may involve basicmethods including Maxam-Gilbert sequencing and chain-terminationmethods, or de novo sequencing methods including shotgun sequencing andbridge PCR, or next-generation methods including polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrentsemiconductor sequencing, HeliScope single molecule sequencing, SMRT®sequencing, and others. Nucleic acid sequencing technologies, includingnext-generation DNA sequencing, have been useful for genomic andproteomic analysis of cell populations.

SUMMARY

Recognized herein is the need for methods, compositions and systems foranalyzing genomic and proteomic information from individual cells or asmall population of cells. Such cells include, but are not limited to,cancer cells, fetal cells, and immune cells involved in immuneresponses. Provided herein are methods, compositions and systems foranalyzing individual cells or a small population of cells, including theanalysis and attribution of nucleic acids and proteins from and to theseindividual cells or cell populations.

In an aspect, the present disclosure provides a method of characterizinga cell. The method comprises (a) providing a partition comprising a celland at least one labelling agent, wherein the at least one labellingagent is (i) capable of binding to a cell surface feature of the celland (ii) is coupled to a reporter oligonucleotide comprising a nucleicacid barcode sequence that permits identification of the at least onelabelling agent, wherein the partition comprises an anchoroligonucleotide that is capable of interacting with the reporteroligonucleotide barcode; (b) in the partition, synthesizing a nucleicacid molecule comprising at least a portion of the nucleic acid barcodesequence or a complement thereof; and (c) subjecting the nucleic acidmolecule to sequencing to identify the labelling agent or the cell.

In some embodiments, in (a), the at least one labelling agent is boundto the cell surface feature. In some embodiments, prior to (a), the atleast one labelling agent is subjected to conditions suitable forbinding the at least one labelling agent to the cell surface feature. Insome embodiments, subjecting the at least one labelling agent to theconditions suitable for binding the at least one labelling agent to thecell surface feature is performed when the cell and the at least onelabelling agent are free from the partition. In some embodiments, priorto (a), the at least one labelling agent is coupled to the reporteroligonucleotide.

In some embodiments, in (b), the reporter oligonucleotide is subjectedto a primer extension reaction that generates the nucleic acid molecule.In some embodiments, the primer extension reaction comprises subjectingthe reporter oligonucleotide to conditions suitable to hybridize theanchor oligonucleotide to the reporter oligonucleotide and extend theanchor oligonucleotide using the reporter oligonucleotide as a template.

In some embodiments, in (b), the anchor oligonucleotide is coupled to abead. In some embodiments, in (b), the anchor oligonucleotide is coupledto a bead and the method further comprises releasing the anchoroligonucleotide from the bead prior to the synthesizing. In someembodiments, the bead is a gel bead. In some embodiments, the releasingcomprises subjecting the bead to a stimulus that degrades the bead. Insome embodiments, the stimulus is a chemical stimulus. In someembodiments, the bead comprises at least about 1,000 copies of theanchor oligonucleotide. In some embodiments, the bead comprises at leastabout 10,000 copies of the anchor oligonucleotide. In some embodiments,the bead comprises at least about 100,000 copies of the anchoroligonucleotide.

In some embodiments, prior to (c), the nucleic acid molecule is releasedfrom the partition. In some embodiments, (c) comprises identifying theat least one labelling agent. In some embodiments, (c) comprisesidentifying the cell surface feature from identifying the at least onelabelling agent. In some embodiments, (c) comprises determining anabundance of the given cell surface feature on the cell. In someembodiments, (c) comprises identifying the cell. In some embodiments,(c) comprises identifying the at least one labelling agent and the cell.

In some embodiments, the reporter oligonucleotide comprises a uniquemolecular identification (UMI) sequence. In some embodiments, the UMIsequence permits identification of the cell. In some embodiments, (c)comprises determining a sequence of the UMI sequence and identifying thecell.

In some embodiments, the partition is a droplet in an emulsion. In someembodiments, the at least one labelling agent is selected from the groupcomprising of an antibody, an antibody fragment, a cell surface receptorbinding molecule, a receptor ligand, a small molecule, a bi-specificantibody, a bi-specific T-cell engager, a T-cell receptor engager, aB-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer,a darpin, a protein scaffold, an antigen, an antigen presenting particleand a major histocompatibility complex (MHC). In some embodiments, thecell surface feature is selected from the group comprising of areceptor, an antigen, a surface protein, a transmembrane protein, acluster of differentiation protein, a protein channel, a protein pump, acarrier protein, a phospholipid, a glycoprotein, a glycolipid, acell-cell interaction protein complex, an antigen-presenting complex, amajor histocompatibility complex, an engineered T-cell receptor, aT-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, and an adherens junction. In some embodiments, the partitioncomprises only one cell.

In some embodiments, the cell is bound to at least one of the at leastone labelling agent. In some embodiments, the at least one of the atleast one labelling agent comprises at least two of the same labellingagent. In some embodiments, the at least one of the at least onelabelling agent comprises at least two different labelling agents. Insome embodiments, the cell is bound to at least about 5 differentlabelling agents. In some embodiments, the cell is bound to at leastabout 10 different labelling agents. In some embodiments, the cell isbound to at least about 50 different labelling agents. In someembodiments, the cell is bound to at least about 100 different labellingagents. In some embodiments, the (c) comprises determining an identityof at least a subset of the different labelling agents.

In some embodiments, the method further comprises (i) liberating nucleicacid from the cell and (ii) subjecting the nucleic acid or a derivativethereof to sequencing. In some embodiments, the nucleic acid isliberated from the cell into the partition.

In an aspect, the present disclosure provides a system forcharacterizing a cell. The system comprises an electronic display screencomprising a user interface that displays a graphical element that isaccessible by a user to execute a protocol to characterize the cell; anda computer processor coupled to the electronic display screen andprogrammed to execute the protocol upon selection of the graphicalelement by the user, which protocol comprises: (a) providing a partitioncomprising a cell and at least one labelling agent, wherein the at leastone labelling agent is (i) capable of binding to a cell surface featureof the cell and (ii) is coupled to a reporter oligonucleotide comprisinga nucleic acid barcode sequence that permits identification of the atleast one labelling agent, wherein the partition comprises an anchoroligonucleotide that is capable of interacting with the reporteroligonucleotide barcode; (b) in the partition, synthesizing a nucleicacid molecule comprising at least a portion of the nucleic acid barcodesequence or a complement thereof; and (c) subjecting the nucleic acidmolecule to sequencing to identify the labelling agent or the cell.

In some embodiments, in protocol (a), the at least one labelling agentis bound to the cell surface feature. In some embodiments, prior toprotocol (a), the at least one labelling agent is subjected toconditions suitable for binding the at least one labelling agent to thecell surface feature. In some embodiments, subjecting the at least onelabelling agent to the conditions suitable for binding the at least onelabelling agent to the cell surface feature is performed when the celland the at least one labelling agent are free from the partition. Insome embodiments, prior to protocol (a), the at least one labellingagent is coupled to the reporter oligonucleotide.

In some embodiments, in protocol (b), the reporter oligonucleotide issubjected to a primer extension reaction that generates the nucleic acidmolecule. In some embodiments, the primer extension reaction comprisessubjecting the reporter oligonucleotide to conditions suitable tohybridize the anchor oligonucleotide to the reporter oligonucleotide andextend the anchor oligonucleotide using the reporter oligonucleotide asa template.

In some embodiments, in protocol (b), the anchor oligonucleotide iscoupled to a bead. In some embodiments, in (b), the anchoroligonucleotide is coupled to a bead and the method further comprisesreleasing the anchor oligonucleotide from the bead prior to thesynthesizing. In some embodiments, the bead is a gel bead. In someembodiments, the releasing comprises subjecting the bead to a stimulusthat degrades the bead. In some embodiments, the stimulus is a chemicalstimulus. In some embodiments, the bead comprises at least about 1,000copies of the anchor oligonucleotide. In some embodiments, the beadcomprises at least about 10,000 copies of the anchor oligonucleotide. Insome embodiments, the bead comprises at least about 100,000 copies ofthe anchor oligonucleotide.

In some embodiments, prior to protocol (c), the nucleic acid molecule isreleased from the partition. In some embodiments, protocol (c) comprisesidentifying the at least one labelling agent. In some embodiments,protocol (c) comprises identifying the cell surface feature fromidentifying the at least one labelling agent. In some embodiments,protocol (c) comprises determining an abundance of the given cellsurface feature on the cell. In some embodiments, protocol (c) comprisesidentifying the cell. In some embodiments, protocol (c) comprisesidentifying the at least one labelling agent and the cell.

In some embodiments, the reporter oligonucleotide comprises a uniquemolecular identification (UMI) sequence. In some embodiments, the UMIsequence permits identification of the cell. In some embodiments,protocol (c) comprises determining a sequence of the UMI sequence andidentifying the cell.

In some embodiments, the partition is a droplet in an emulsion. In someembodiments, the at least one labelling agent is selected from the groupcomprising of an antibody, an antibody fragment, a cell surface receptorbinding molecule, a receptor ligand, a small molecule, a bi-specificantibody, a bi-specific T-cell engager, a T-cell receptor engager, aB-cell receptor engager, a pro-body, an aptamer, a monobody, an affimer,a darpin, a protein scaffold, an antigen, an antigen presenting particleand a major histocompatibility complex (MHC). In some embodiments, thecell surface feature is selected from the group comprising of areceptor, an antigen, a surface protein, a transmembrane protein, acluster of differentiation protein, a protein channel, a protein pump, acarrier protein, a phospholipid, a glycoprotein, a glycolipid, acell-cell interaction protein complex, an antigen-presenting complex, amajor histocompatibility complex, an engineered T-cell receptor, aT-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, and an adherens junction. In some embodiments, the partitioncomprises only one cell.

In some embodiments, the cell is bound to at least one of the at leastone labelling agent. In some embodiments, the at least one of the atleast one labelling agent comprises at least two of the same labellingagent. In some embodiments, the at least one of the at least onelabelling agent comprises at least two different labelling agents. Insome embodiments, the cell is bound to at least about 5 differentlabelling agents. In some embodiments, the cell is bound to at leastabout 10 different labelling agents. In some embodiments, the cell isbound to at least about 50 different labelling agents. In someembodiments, the cell is bound to at least about 100 different labellingagents. In some embodiments, protocol (c) comprises determining anidentity of at least a subset of the different labelling agents.

In some embodiments, protocol comprises (i) liberating nucleic acid fromthe cell and (ii) subjecting the nucleic acid or a derivative thereof tosequencing. In some embodiments, the nucleic acid is liberated from thecell into the partition.

An additional aspect of the disclosure provides a method for analytecharacterization. The method includes: (a) providing a plurality ofpartitions, where a given partition of the plurality of partitionscomprises a plurality of barcode molecules and a plurality of analytes.In some cases, the plurality of barcode molecules comprises at least1,000 barcode molecules. In addition, (i) a first individual barcodemolecule of the plurality of barcode molecules can comprise a firstnucleic acid barcode sequence that is capable of coupling to a firstanalyte of the plurality of analytes, and (ii) a second individualbarcode molecule of the plurality of barcoded molecules can comprise asecond nucleic acid barcode sequence that is capable of coupling to asecond analyte of the plurality of analytes where the first analyte andthe second analyte are different types of analytes. The method alsoincludes (b) in the given partition, (i) synthesizing a first nucleicacid molecule comprising at least a portion of the first nucleic acidbarcode sequence or complement thereof, and (ii) synthesizing a secondnucleic acid molecule comprising at least a portion of the secondnucleic acid barcode sequence or complement thereof; and (c) removingthe first nucleic acid molecule and the second nucleic acid moleculefrom the given partition.

In some embodiments, the method further comprises subjecting the firstnucleic acid molecule and the second nucleic acid molecule, or aderivative of the first nucleic acid molecule and/or the second nucleicacid molecule, to sequencing to characterize the first analyte and/orthe second analyte. In some embodiments, the method further comprisesrepeating (a)-(c) based on a characterization of the first analyte orthe second analyte from the sequencing. In some embodiments, the methodfurther comprises selecting the first analyte or the second analytebased on a characterization of the first analyte or the second analyteobtained from the sequencing or a subsequent sequencing upon repeating(a)-(c).

In some embodiments, (b) further comprises: (1) synthesizing the firstnucleic acid molecule comprising at least a portion of the first nucleicacid barcode sequence or complement thereof, and (2) synthesizing thesecond nucleic acid molecule comprising at least a portion of the secondnucleic acid barcode sequence or complement thereof.

In some embodiments, the first analyte is a nucleic acid molecule, suchas genomic deoxyribonucleic acid (gDNA) or messenger RNA (mRNA).

In some embodiments, the first analyte is a labelling agent capable ofcoupling to a cell surface feature of a cell. In some embodiments, thefirst individual barcode molecule or the second individual barcodemolecule is capable of coupling to the labelling agent via a thirdnucleic acid molecule coupled to the labelling agent. In someembodiments, the cell surface feature is a receptor, an antigen, or aprotein. In some embodiments, the labelling agent is an antibody, anantibody fragment or a major histocompatibility complex (MHC). In someembodiments, the given partition comprises the cell or one or morecomponents of the cell. In some embodiments, the given partitioncomprises a single cell. In some embodiments, the first nucleic acidmolecule or the second nucleic molecule comprises a third barcodesequence. In some embodiments, the third barcode sequence is derivedfrom a third nucleic acid molecule. In some embodiments, the thirdnucleic acid molecule is coupled to a labelling agent capable of bindingto a cell surface feature of a cell.

In some embodiments, the first analyte and second analyte are differenttypes of nucleic acid molecules. In some embodiments, the first analyteis a ribonucleic acid molecule and the second analyte is adeoxyribonucleic acid molecule. In some embodiments, (i) the firstindividual barcode molecule comprises a first priming sequence capableof hybridizing to the first analyte; or (ii) the second individualbarcode molecule comprises a second priming sequence capable ofhybridizing to the second analyte. In some embodiments, the firstbarcode molecule or the second barcode molecule comprises a uniquemolecular identification (UMI) sequence.

In some embodiments, the first analyte is a nucleic acid molecule andthe second analyte is a labelling agent capable of coupling to a cellsurface feature. In some embodiments, the first analyte is a messengerribonucleic acid molecule. In some embodiments, (i) the first individualbarcode molecule comprises a first priming sequence capable ofhybridizing to the first analyte; or (ii) the second individual barcodemolecule comprises a second priming sequence capable of hybridizing tothe labelling agent. In some embodiments, the labelling agent is anantibody, an antibody fragment, or a major histocompatibility complex(MHC). In some embodiments, the cell surface feature is selected fromthe group consisting of a receptor, an antigen, or a protein.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence or complement thereof that encodes at leasta portion of a V(D)J sequence of an immune cell receptor. In someembodiments, the nucleic acid molecule is a messenger ribonucleic acid.In some embodiments, the nucleic acid molecule is complementary DNA(cDNA) derived from reverse transcription of an mRNA encoding the atleast a portion of the V(D)J sequence.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence that is capable of functioning as acomponent of a gene editing reaction. In some embodiments, the geneediting reaction comprises clustered regularly interspaced shortpalindromic repeats (CRISPR)-based gene editing.

In some embodiments, at least one of the first individual barcodemolecule and the second individual barcode molecule is coupled to abead, such as a gel bead. The bead can be degradable. In someembodiments, the method further comprises, after (a), releasing thefirst individual barcode molecule or the second individual barcode fromthe bead. In some embodiments, the given partition further comprises anagent capable of releasing the first individual barcode molecule or thesecond individual barcode from the bead.

In some embodiments, the given partition selected is a droplet among aplurality of droplets or a well among a plurality of wells. In someembodiments, the first nucleic acid barcode sequence and the secondnucleic barcode sequence are identical. In some embodiments, the methodfurther comprises performing one or more reactions subsequent toremoving the first nucleic acid molecule and the second nucleic acidmolecule from the given partition.

Another aspect of the disclosure provides a composition forcharacterizing a plurality of analytes. The composition comprises apartition comprising a plurality of barcode molecules and the pluralityof analytes. The plurality of barcode molecules can comprise at least1,000 barcode molecules. In addition, (i) a first individual barcodemolecule of the plurality of barcode molecules can comprise a firstnucleic acid barcode sequence that is capable of coupling to a firstanalyte of the plurality of analytes; and (ii) a second individualbarcode molecule of the plurality of barcoded molecules can comprise asecond nucleic acid barcode sequence that is capable of coupling to asecond analyte of the plurality of analytes, where the first analyte andthe second analyte are different types of analytes.

In some embodiments, the first analyte is a nucleic acid molecule, suchas genomic deoxyribonucleic acid (gDNA) or is messenger RNA (mRNA).

In some embodiments, the first analyte is a labelling agent capable ofcoupling to a cell surface feature of a cell. In some embodiments, thefirst individual barcode molecule or the second individual barcodemolecule is capable of coupling to the labelling agent via a thirdnucleic acid molecule coupled to the labelling agent. In someembodiments, the cell surface feature is a receptor, an antigen, or aprotein. In some embodiments, the labelling agent is an antibody, anantibody fragment, an antibody fragment, or a major histocompatibilitycomplex (MHC). In some embodiments, the partition comprises the cell orone or more components of the cell. In some embodiments, the partitioncomprises a single cell. In some embodiments, the first nucleic acidmolecule or the second nucleic molecule comprises a third barcodesequence. In some embodiments, the third barcode sequence is derivedfrom a third nucleic acid molecule. In some embodiments, the thirdnucleic acid molecule is coupled to a labelling agent capable of bindingto a cell surface feature of a cell.

In some embodiments, the first analyte and second analyte are differenttypes nucleic acid molecules. In some embodiments, the first analyte isa ribonucleic acid molecule and the second analyte is a deoxyribonucleicacid molecule. In some embodiments, (i) the first individual barcodemolecule comprises a first priming sequence capable of hybridizing tothe first analyte; or (ii) the second individual barcode moleculecomprises a second priming sequence capable of hybridizing to the secondanalyte. In some embodiments, the first barcode molecule or the secondbarcode molecule comprises a unique molecular identification (UMI)sequence.

In some embodiments, the first analyte is a nucleic acid molecule andthe second analyte is a labelling agent capable of coupling to a cellsurface feature. In some embodiments, the first analyte is a ribonucleicacid molecule. In some embodiments, (i) the first individual barcodemolecule comprises a first priming sequence capable of hybridizing tothe first analyte; or (ii) the second individual barcode moleculecomprises a second priming sequence capable of hybridizing to a thirdnucleic acid molecule coupled to the labelling agent. In someembodiments, the labelling agent is an antibody, an antibody fragment,or a major histocompatibility complex (MHC). In some embodiments, thecell surface feature is a receptor, an antigen, or a protein.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence or complement thereof that encodes at leasta portion of a V(D)J sequence of an immune cell receptor. In someembodiments, the nucleic acid sequence is a ribonucleic acid molecule.In some embodiments, the nucleic acid molecule comprises a nucleic acidsequence that is complementary DNA (cDNA) derived from reversetranscription of an mRNA encoding the at least a portion of the V(D)Jsequence.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence that is capable of functioning as acomponent of a gene editing reaction. In some embodiments, the geneediting reaction comprises clustered regularly interspaced shortpalindromic repeats (CRISPR)-based gene editing. In some embodiments, atleast one of the first individual barcode molecule and the secondindividual barcode molecule is coupled to a bead, such as a gel bead.The bead may be degradable. In some embodiments, the given partitionfurther comprises an agent capable of releasing the first individualbarcode molecule or the second individual barcode from the bead. In someembodiments, the given partition is a droplet among a plurality ofdroplets or a well among a plurality of wells. In some embodiments, thefirst nucleic acid barcode sequence and the second nucleic barcodesequence are identical.

An additional aspect of the disclosure provides a system forcharacterizing a plurality of analytes. The system comprises apartitioning unit for providing a partition comprising a plurality ofbarcode molecules and the plurality of analytes, where: (i) a firstindividual barcode molecule of the plurality of barcode moleculescomprises a first nucleic acid barcode sequence and is capable ofcoupling to a first analyte of the plurality of analytes; and (ii) asecond individual barcode molecule of the plurality of barcode moleculescomprises a second nucleic acid barcode sequence and is capable ofcoupling to a second analyte of the plurality of analytes, where thefirst analyte and the second analyte are different types of analytes.The system also includes a controller coupled to the partitioning unit,where the controller is programmed to (i) direct the partitioning unitto provide the partition; (ii) subject the partition to conditions thatare sufficient to: (1) synthesize a first nucleic acid moleculecomprising at least a portion of the first nucleic acid barcode sequenceor complement thereof; and (2) synthesize a second nucleic acid moleculecomprising at least a portion of the second nucleic acid barcodesequence or complement thereof, where sequencing of the first nucleicacid molecule and the second nucleic acid molecule, or derivativesthereof, characterizes the first analyte or the second analyte.

In some embodiments, the partitioning unit comprises a plurality ofchannels. In some embodiments, the partitioning unit further comprisesat least one channel junction, where the at least one channel junctionis configured to provide the partition. In some embodiments, the systemalso includes (i) a first channel fluidically connected to the at leastone channel junction and configured to provide a first fluid to the atleast one channel junction; (ii) and a second channel fluidicallyconnected to the at least one channel junction and configured to providea second fluid, immiscible with the first fluid, to the at least onechannel junction.

In some embodiments, the first analyte is a nucleic acid molecule, suchas genomic deoxyribonucleic acid (gDNA) or messenger RNA (mRNA).

In some embodiments, the first analyte is a labelling agent capable ofcoupling to a cell surface feature of a cell. In some embodiments, thefirst individual barcode molecule or the second individual barcodemolecule is capable of coupling to the labelling agent via a thirdnucleic acid molecule coupled to the labelling agent. In someembodiments, the cell surface feature is a receptor, an antigen, or aprotein. In some embodiments, the labelling agent is an antibody, anantibody fragment, or a major histocompatibility complex (MHC). In someembodiments, the partition comprises the cell or one or more componentsof the cell. In some embodiments, the partition comprises a single cell.In some embodiments, the first nucleic acid molecule or the secondnucleic molecule comprises a third barcode sequence. In someembodiments, the third barcode sequence is derived from a third nucleicacid molecule. In some embodiments, the third nucleic acid molecule iscoupled to a labelling agent capable of binding to a cell surfacefeature of a cell.

In some embodiments, the first analyte and second analyte are differenttypes nucleic acid molecules. In some embodiments, the first analyte isa ribonucleic acid molecule and the second analyte is a deoxyribonucleicacid molecule. In some embodiments, (i) the first individual barcodemolecule comprises a first priming sequence capable of hybridizing tothe first analyte; or (ii) the second individual barcode moleculecomprises a second priming sequence capable of hybridizing to the secondanalyte. In some embodiments, the first barcode molecule or the secondbarcode molecule comprises a unique molecular identification (UMI)sequence.

In some embodiments, the first analyte is a nucleic acid molecule andthe second analyte is a labelling agent capable of coupling to a cellsurface feature. In some embodiments, the first analyte is a ribonucleicacid molecule. In some embodiments, (i) the first individual barcodemolecule comprises a first priming sequence capable of hybridizing tothe first analyte; or (ii) the second individual barcode moleculecomprises a second priming sequence capable of hybridizing to a thirdnucleic acid molecule coupled to the labelling agent. In someembodiments, the labelling agent is an antibody, an antibody fragment,or a major histocompatibility complex (MHC). In some embodiments, thecell surface feature is a receptor, an antigen, or a protein.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence or complement thereof that encodes at leasta portion of a V(D)J sequence of an immune cell receptor. In someembodiments, the nucleic acid sequence is a messenger ribonucleic acidmolecule. In some embodiments, the nucleic acid molecule iscomplementary DNA (cDNA) derived from reverse transcription of an mRNAencoding the at least a portion of the V(D)J sequence.

In some embodiments, the first analyte comprises a nucleic acid moleculewith a nucleic acid sequence that is capable of functioning as acomponent of a gene editing reaction. In some embodiments, the geneediting reaction comprises clustered regularly interspaced shortpalindromic repeats (CRISPR)-based gene editing.

In some embodiments, at least one of the first individual barcodemolecule and the second individual barcode molecule is coupled to abead, such as a gel bead. The bead may be degradable. In someembodiments, the partition further comprises an agent capable ofreleasing the first individual barcode molecule or the second individualbarcode from the bead. In some embodiments, the partition is a dropletamong a plurality of droplets or a well among a plurality of wells. Insome embodiments, the nucleic acid barcode sequence and the secondnucleic barcode sequence are identical. In some embodiments, thepartition comprises at least 1,000 barcode molecules.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings (also “Figure” and “FIG.” herein), of which:

FIG. 1 schematically illustrates a microfluidic channel structure forpartitioning individual or small groups of cells;

FIG. 2 schematically illustrates a microfluidic channel structure forco-partitioning cells and microcapsules (e.g., beads) comprisingadditional reagents;

FIGS. 3A-3F schematically illustrate an example process foramplification and barcoding of cell's nucleic acids;

FIG. 4 provides a schematic illustration of use of barcoding of cell'snucleic acids in attributing sequence data to individual cells or groupsof cells for use in their characterization;

FIG. 5 provides a schematic illustration of cells associated withlabeled cell-binding ligands;

FIG. 6 provides a schematic illustration of an example workflow forperforming RNA analysis using the methods described herein;

FIG. 7 provides a schematic illustration of an example barcodedoligonucleotide structure for use in analysis of ribonucleic (RNA) usingthe methods described herein;

FIG. 8 provides an image of individual cells co-partitioned along withindividual barcode bearing beads;

FIGS. 9A-9E provide schematic illustrations of example barcodedoligonucleotide structures for use in analysis of RNA and exampleoperations for performing RNA analysis;

FIG. 10 provides a schematic illustration of example barcodedoligonucleotide structure for use in example analysis of RNA and use ofa sequence for in vitro transcription;

FIG. 11 provides a schematic illustration of an example barcodedoligonucleotide structure for use in analysis of RNA and exampleoperations for performing RNA analysis;

FIGS. 12A-12B provide schematic illustrations of example barcodedoligonucleotide structure for use in analysis of RNA;

FIGS. 13A-13C provide illustrations of example yields from templateswitch reverse transcription and PCR in partitions;

FIGS. 14A-14B provide illustrations of example yields from reversetranscription and cDNA amplification in partitions with various cellnumbers;

FIG. 15 provides an illustration of example yields from cDNA synthesisand real-time quantitative PCR at various input cell concentrations andalso the effect of varying primer concentration on yield at a fixed cellinput concentration;

FIG. 16 provides an illustration of example yields from in vitrotranscription;

FIG. 17 shows an example computer control system that is programmed orotherwise configured to implement methods provided herein;

FIG. 18 provides a schematic illustration of an example barcodedoligonucleotide structure;

FIG. 19 shows example operations for performing RNA analysis;

FIG. 20 shows a method for characterizing a cell, according toembodiments;

FIG. 21 shows an oligonucleotide with modifications that may preventextension by a polymerase;

FIG. 22 shows oligonucleotides comprising a U-excising element;

FIG. 23A shows a bead coupled with an oligonucleotide comprising atarget-specific primer and oligonucleotides with poly-T primers; FIG.23B shows a bead coupled with a plurality of oligonucleotides, each ofwhich comprises a target-specific primer; FIG. 23C shows a bead coupledwith a plurality of oligonucleotides, each of which comprises atarget-specific primer and a plurality of oligonucleotides, each ofwhich comprises a poly-T primer;

FIG. 24 shows a bead coupled with a plurality of oligonucleotides, eachof which comprises a target-specific primer and a plurality ofoligonucleotides, each of which comprises a random N-mer primer fortotal RNA;

FIG. 25 shows oligonucleotides comprising adapters and assay primers;

FIG. 26 shows an oligonucleotide with an adapter comprising a switcholigo;

FIG. 27A shows oligonucleotides with backbones comprising P7 and R2sequences and poly-T primers. FIG. 27B shows y oligonucleotides withbackbones comprising R1 sequences and poly-T primers. FIG. 27C showsoligonucleotides with P5, R1, and R2 sequences and poly-T primers. FIG.27D shows oligonucleotides with R1 sequences and random N-mer primers.

FIG. 28 shows a workflow for conjugating a DNA barcode on an antibodyusing an antibody-binding protein;

FIG. 29 demonstrates swelling conditions and de-swelling conditions inthe process of making gel beads with magnetic particles;

FIG. 30 shows a unit cell comprising a scaffold and liquid immediatelysurrounding the scaffold;

FIG. 31 shows a microcapsule with a barcoded magnetic particleentrapped;

FIG. 32 shows a method for parallel sequencing DNA molecules and RNAmolecules in a cell;

FIG. 33 shows various approaches for making antibody-reporteroligonucleotide conjugates;

FIG. 34 shows an antibody-reporter oligonucleotide conjugation;

FIGS. 35A-35C show a method for analyzing mRNA molecules and proteinsfrom a single cell;

FIG. 36A shows a relationship between a diameter of a gel bead and aregent inside the gel bead; FIG. 36B shows the relationship between thediameter of a gel bead and the number of droplets with more than onecell;

FIG. 37 shows analysis results of the CD3 protein-single-stranded DNA(ssDNA) conjugate;

FIG. 38 shows the fluorescence signals from the cells bound by labeledantibodies;

FIG. 39A shows an approach for conjugating an oligonucleotide with anantibody; FIG. 39B shows analysis results of barcoded antibodies;

FIG. 40 shows a conjugate of a functionalized antibody-binding proteinand a functionalized oligonucleotide;

FIG. 41 shows a relationship between a degree of dibenzocyclooctyne(DBCO) incorporation and input dibenzocyclooctyne-N-hydroxysuccinimidylester (DBCO-NHS) concentrations;

FIG. 42 shows an example relationship between the degree of conjugationand oligonucleotide equivalence;

FIG. 43 shows fluorescence signals of labeled cells measured by flowcytometry;

FIG. 44 shows a method for producing a bead coupled witholigonucleotides with different primer sequences;

FIG. 45A shows a bead coupled with a plurality of oligonucleotides. FIG.45B shows results from gel electrophoresis analysis of beads. On thebeads, 0%, 5%, 15%, or 25% of coupled oligonucleotides contain antibodytarget primers;

FIGS. 46A-46C schematically depict components of example multi-assayschemes described herein;

FIG. 47 depicts data obtained from an example experiment described inExample XI;

FIG. 48 depicts data obtained from an example experiment described inExample XI;

FIGS. 49A and 49B depict data obtained from an example experimentdescribed in Example XI;

FIG. 50A schematically depicts an example bead comprisingoligonucleotides having two different functional sequences;

FIGS. 50B and 50C schematically depict example sequences that can becoupled to a bead;

FIG. 51A depicts sequences used in an example experiment described inExample XII;

FIG. 51B graphically depicts data from an example experiment describedin Example XII;

FIG. 52A depicts data obtained from an example experiment described inExample XIII;

FIG. 52B provides schematically depicts example extension schemes tolink barcodes;

FIGS. 53A and 53B provide data obtained from an example experimentdescribed in Example XIII;

FIGS. 54 and 55 provide data obtained from example experiments describedin Example XIV; and

FIGS. 56A-C schematically depict an example barcoding scheme thatincludes major histocompatibility complexes.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and describedherein, it will be obvious to those skilled in the art that suchembodiments are provided by way of example only. Numerous variations,changes, and substitutions may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

Where values are described as ranges, it will be understood that suchdisclosure includes the disclosure of all possible sub-ranges withinsuch ranges, as well as specific numerical values that fall within suchranges irrespective of whether a specific numerical value or specificsub-range is expressly stated.

The term “barcode,” as used herein, generally refers to a label, oridentifier, that can be part of an analyte to convey information aboutthe analyte. A barcode can be a tag attached to an analyte (e.g.,nucleic acid molecule) or a combination of the tag in addition to anendogenous characteristic of the analyte (e.g., size of the analyte orend sequence(s)). The barcode may be unique. Barcodes can have a varietyof different formats, for example, barcodes can include: polynucleotidebarcodes; random nucleic acid and/or amino acid sequences; and syntheticnucleic acid and/or amino acid sequences. A barcode can be attached toan analyte in a reversible or irreversible manner. A barcode can beadded to, for example, a fragment of a deoxyribonucleic acid (DNA) orribonucleic acid (RNA) sample before, during, and/or after sequencing ofthe sample. Barcodes can allow for identification and/or quantificationof individual sequencing-reads in real time.

The term “subject,” as used herein, generally refers to an animal, suchas a mammalian species (e.g., human) or avian (e.g., bird) species, orother organism, such as a plant. The subject can be a vertebrate, amammal, a mouse, a primate, a simian or a human. Animals may include,but are not limited to, farm animals, sport animals, and pets. A subjectcan be a healthy individual, an individual that has or is suspected ofhaving a disease or a pre-disposition to the disease, or an individualthat is in need of therapy or suspected of needing therapy. A subjectcan be a patient.

The term “genome,” as used herein, generally refers to an entirety of asubject's hereditary information. A genome can be encoded either in DNAor in RNA. A genome can comprise coding regions that code for proteinsas well as non-coding regions. A genome can include the sequence of allchromosomes together in an organism. For example, the human genome has atotal of 46 chromosomes. The sequence of all of these together mayconstitute a human genome.

The terms “adaptor(s)”, “adapter(s)” and “tag(s)” may be usedsynonymously. An adaptor or tag can be coupled to a polynucleotidesequence to be “tagged” by any approach including ligation,hybridization, or other approaches.

The term “sequencing,” as used herein, generally refers to methods andtechnologies for determining the sequence of nucleotide bases in one ormore polynucleotides. The polynucleotides can be, for example,deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), includingvariants or derivatives thereof (e.g., single stranded DNA). Sequencingcan be performed by various systems currently available, such as,without limitation, a sequencing system by Illumina, PacificBiosciences, Oxford Nanopore, or Life Technologies (Ion Torrent). Suchdevices may provide a plurality of raw genetic data corresponding to thegenetic information of a subject (e.g., human), as generated by thedevice from a sample provided by the subject. In some situations,systems and methods provided herein may be used with proteomicinformation.

The term “variant,” as used herein, generally refers to a geneticvariant, such as a nucleic acid molecule comprising a polymorphism. Avariant can be a structural variant or copy number variant, which can begenomic variants that are larger than single nucleotide variants orshort indels. A variant can be an alteration or polymorphism in anucleic acid sample or genome of a subject. Single nucleotidepolymorphisms (SNPs) are a form of polymorphisms. Polymorphisms caninclude single nucleotide variations (SNVs), insertions, deletions,repeats, small insertions, small deletions, small repeats, structuralvariant junctions, variable length tandem repeats, and/or flankingsequences. Copy number variants (CNVs), transversions and otherrearrangements are also forms of genetic variation. A genomic alterationmay be a base change, insertion, deletion, repeat, copy numbervariation, or transversion.

The term “bead,” as used herein, generally refers to a particle. Thebead may be a solid or semi-solid particle. The bead may be a gel. Thebead may be formed of a polymeric material. The bead may be magnetic ornon-magnetic.

The term “sample,” as used herein, generally refers to a biologicalsample of a subject. The sample may be a tissue sample, such as abiopsy, core biopsy, needle aspirate, or fine needle aspirate. Thesample may be a fluid sample, such as a blood sample, urine sample, orsaliva sample. The sample may be a skin sample. The sample may be acheek swab. The sample may be a plasma or serum sample. The sample maybe a cell-free (or cell free) sample. A cell-free sample may includeextracellular polynucleotides. Extracellular polynucleotides may beisolated from a bodily sample that may be selected from a groupconsisting of blood, plasma, serum, urine, saliva, mucosal excretions,sputum, stool and tears.

The term “nucleic acid,” as used herein, generally refers to a monomericor polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs or variants thereof.A nucleic acid molecule may include one or more unmodified or modifiednucleotides. Nucleic acid may have any three dimensional structure, andmay perform any function. The following are non-limiting examples ofnucleic acids: ribonucleic acid (RNA), deoxyribonucleic acid (DNA),coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer ribonucleic acid (RNA), ribosomal RNA, short interfering RNA(siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes,complementary deoxyribonucleic acid (cDNA), recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. Nucleic acid may comprise one or more modified nucleotides,such as methylated nucleotides and nucleotide analogs, such as peptidenucleic acid (PNA), Morpholino and locked nucleic acid (LNA), glycolnucleic acid (GNA), threose nucleic acid (TNA), 2′-fluoro, 2′-OMe, andphosphorothiolated DNA. A nucleic acid may include one or more subunitsselected from adenosine (A), cytosine (C), guanine (G), thymine (T) anduracil (U), or variants thereof. In some examples, a nucleic acid is DNAor RNA, or derivatives thereof. A nucleic acid may be single-stranded ordouble stranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which may include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (i.e., A or G, or variantor analogs thereof) or a pyrimidine (i.e., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

The term “analyte,” as used herein, generally refers to a substance orone or more constituents thereof that is for identification, such asdetection (e.g., detection via sequencing). Examples of analytesinclude, without limitation, DNA, RNA, a labelling agent, antibody, andprotein. An analyte may be a cell or one or more constituents of a cell.

Analytes may be of different types. In some examples, in a plurality ofanalytes, a given analyte is of a different structural or functionalclass from other analytes of the plurality. Examples of different typesof analytes include DNA and RNA; a nucleic acid molecule and a labellingagent; a transcript and genomic nucleic acid; a plurality of nucleicacid molecules, where each nucleic acid molecule has a differentfunction, such as a different cellular function. A sample may have aplurality of analytes of different types, such as a mixture of DNA andRNA molecules, or a mixture of nucleic acid molecules and labellingagents. In some cases, different types of analytes do not includelabelling agents directed to separate cell surface features of a cell.

Nucleic acid sequencing technologies have yielded substantial results insequencing biological materials, including providing substantialsequence information on individual organisms, and relatively purebiological samples. However, these systems have traditionally not beeneffective at being able to identify and characterize cells at the singlecell level.

Nucleic acid sequencing technologies may derive the nucleic acids thatthey sequence from collections of cells obtained from tissue or othersamples, such as biological fluids (e.g., blood, plasma, etc). The cellscan be processed (e.g., all together in an ensemble approach) to extractthe genetic material that represents an average of the population ofcells, which can then be processed into sequencing ready DNA librariesthat are configured for a given sequencing technology. Although oftendiscussed in terms of DNA or nucleic acids, the nucleic acids derivedfrom the cells may include DNA, or RNA, including, e.g., mRNA, totalRNA, or the like, that may be processed to produce cDNA for sequencing.

In addition to the inability to attribute characteristics to particularsubsets of cells or individual cells, such ensemble sample preparationmethods can be, from the outset, predisposed to primarily identifyingand characterizing the majority constituents in the sample of cells, andmay not be designed to pick out the minority constituents, e.g., geneticor proteomic material contributed by one cell, a few cells, or a smallpercentage of total cells in the sample. Likewise, where analyzingexpression levels, e.g., of mRNA or cell surface proteins, an ensembleapproach can be predisposed to presenting potentially inaccurate datafrom cell populations that are non-homogeneous in terms of expressionlevels. In some cases, where expression is high in a small minority ofthe cells in an analyzed population, and absent in the majority of thecells of the population, an ensemble method may indicate low levelexpression for the entire population.

These inaccuracies can be further magnified through processingoperations used in generating the sequencing libraries from thesesamples. Some next generation sequencing technologies (e.g., massivelyparallel sequencing) may rely upon the geometric amplification ofnucleic acid fragments, such as via polymerase chain reaction, in orderto produce sufficient DNA for the sequencing library. However, suchamplification can be biased toward amplification of majorityconstituents in a sample, and may not preserve the starting ratios ofsuch minority and majority components. While some of these difficultiesmay be addressed by utilizing different sequencing systems, such assingle molecule systems that do not require amplification, the singlemolecule systems, as well as the ensemble sequencing methods of othernext generation sequencing (NGS) systems, can also have large input DNArequirements. Some single molecule sequencing systems, for example, canhave sample input DNA requirements of from 500 nanograms (ng) to upwardsof 10 micrograms (m), which may not be obtainable from individual cellsor small subpopulations of cells. Likewise, other NGS systems can beoptimized for starting amounts of sample DNA in the sample of fromapproximately 50 nanograms (ng) to about 1 microgram (m). Startingamounts of DNA may be at least about 1 ng, 10 ng, 20 ng, 30 ng, 40 ng,50 ng, 100 ng, 500 ng, 1 μs, 10 μs, or 100 μg.

Disclosed herein are methods and systems for characterizing surfacefeatures, proteins, and nucleic acids of small populations of cells, andin some cases, for characterizing surface features, proteins, andnucleic acids of individual cells. The methods described herein maycompartmentalize the analysis of individual cells or small populationsof cells, including e.g., cell surface features, proteins, and nucleicacids of individual cells or small groups of cells, and then allow thatanalysis to be attributed back to the individual cell or small group ofcells from which the cell surface features, proteins, and nucleic acidswere derived. This can be accomplished regardless of whether the cellpopulation represents a 50/50 mix of cell types, a 90/10 mix of celltypes, or virtually any ratio of cell types, as well as a completeheterogeneous mix of different cell types, or any mixture between these.Differing cell types may include cells from different tissue types of anindividual or the same tissue type from different individuals, orbiological organisms such as microorganisms from differing genera,species, strains, variants, or any combination of any or all of theforegoing. For example, differing cell types may include normal andtumor tissue from an individual, various cell types obtained from ahuman subject such as a variety of immune cells (e.g., B cells, T cells,and the like), multiple different bacterial species, strains and/orvariants from environmental, forensic, microbiome or other samples, orany of a variety of other mixtures of cell types.

In one aspect, the methods and systems described herein provide for thecompartmentalization, depositing or partitioning of the nucleic acidcontents of individual cells from a sample material containing cells,into discrete compartments or partitions (referred to interchangeablyherein as partitions), where each partition maintains separation of itsown contents from the contents of other partitions. In another aspect,the methods and system described herein provide for thecompartmentalization, depositing or partitioning of individual cellsfrom a sample material containing cells, into discrete partitions, whereeach partition maintains separation of its own contents from thecontents of other partitions. In another aspect, the methods and systemdescribed herein provide for the compartmentalization, depositing orpartitioning of individual cells from a sample material containing cellsafter at least one labelling agent has been bound to a cell surfacefeature of the cell, into discrete partitions, where each partitionmaintains separation of its own contents from the contents of otherpartitions. Unique identifiers, e.g., barcodes, may be previously,subsequently or concurrently delivered to the partitions that hold thecompartmentalized or partitioned cells, in order to allow for the laterattribution of the characteristics of the individual cells to theparticular compartment. Further, unique identifiers, e.g., barcodes, maybe coupled to labelling agents and previously, subsequently orconcurrently delivered to the partitions that hold the compartmentalizedor partitioned cells, in order to allow for the later attribution of thecharacteristics of the individual cells to the particular compartment.Barcodes may be delivered, for example on an oligonucleotide, to apartition via any suitable mechanism.

In some embodiments, barcoded oligonucleotides are delivered to apartition via a microcapsule. In some cases, barcoded oligonucleotidesare initially associated with the microcapsule and then released fromthe microcapsule upon application of a stimulus which allows theoligonucleotides to dissociate or to be released from the microcapsule.In some embodiments, anchor oligonucleotides are delivered to apartition via a microcapsule. In some cases, anchor oligonucleotides areinitially associated with the microcapsule and then released from themicrocapsule upon application of a stimulus which allows the anchoroligonucleotides to dissociate or to be released from the microcapsule.

A microcapsule may be or may include a solid support or solid particlesuch as a bead. A solid support or a solid particle may be a bead. Amicrocapsule may be a droplet. A microcapsule, in some embodiments, maybe or may comprise a bead. In some embodiments, a bead may be porous,non-porous, solid, semi-solid, semi-fluidic, or fluidic. In someembodiments, a bead may be dissolvable, disruptable, or degradable. Insome cases, a bead may not be degradable. In some embodiments, the beadmay be a gel bead. A gel bead may be a hydrogel bead. A gel bead may beformed from molecular precursors, such as a polymeric or monomericspecies. A semi-solid bead may be a liposomal bead. Solid beads maycomprise metals including iron oxide, gold, and silver. In some cases,the beads may be silica beads. In some cases, the beads may be rigid. Insome cases, the beads may be flexible and/or compressible.

In some embodiments, the bead may contain molecular precursors (e.g.,monomers or polymers), which may form a polymer network viapolymerization of the precursors. In some cases, a precursor may be analready polymerized species capable of undergoing further polymerizationvia, for example, a chemical cross-linkage. In some cases, a precursorcomprises one or more of an acrylamide or a methacrylamide monomer,oligomer, or polymer. In some cases, the bead may comprise prepolymers,which are oligomers capable of further polymerization. For example,polyurethane beads may be prepared using prepolymers. In some cases, thebead may contain individual polymers that may be further polymerizedtogether. In some cases, beads may be generated via polymerization ofdifferent precursors, such that they comprise mixed polymers,co-polymers, and/or block co-polymers.

A bead may comprise natural and/or synthetic materials. For example, apolymer can be a natural polymer or a synthetic polymer. In some cases,a bead may comprise both natural and synthetic polymers. Examples ofnatural polymers include proteins and sugars such as deoxyribonucleicacid, rubber, cellulose, starch (e.g., amylose, amylopectin), proteins,enzymes, polysaccharides, silks, polyhydroxyalkanoates, chitosan,dextran, collagen, carrageenan, ispaghula, acacia, agar, gelatin,shellac, sterculia gum, xanthan gum, Corn sugar gum, guar gum, gumkaraya, agarose, alginic acid, alginate, or natural polymers thereof.Examples of synthetic polymers include acrylics, nylons, silicones,spandex, viscose rayon, polycarboxylic acids, polyvinyl acetate,polyacrylamide, polyacrylate, polyethylene glycol, polyurethanes,polylactic acid, silica, polystyrene, polyacrylonitrile, polybutadiene,polycarbonate, polyethylene, polyethylene terephthalate,poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethyleneterephthalate), polyethylene, polyisobutylene, poly(methylmethacrylate), poly(oxymethylene), polyformaldehyde, polypropylene,polystyrene, poly(tetrafluoroethylene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene dichloride),poly(vinylidene difluoride), poly(vinyl fluoride) and combinations(e.g., co-polymers) thereof. Beads may also be formed from materialsother than polymers, including lipids, micelles, ceramics,glass-ceramics, material composites, metals, other inorganic materials,and others.

In some cases, a chemical cross-linker may be a precursor used tocross-link monomers during polymerization of the monomers and/or may beused to attach oligonucleotides (e.g., barcoded oligonucleotides) to thebead. In some cases, polymers may be further polymerized with across-linker species or other type of monomer to generate a furtherpolymeric network. Non-limiting examples of chemical cross-linkers (alsoreferred to as a “crosslinker” or a “crosslinker agent” herein) includecystamine, gluteraldehyde, dimethyl suberimidate, N-Hydroxysuccinimidecrosslinker BS3, formaldehyde, carbodiimide (EDC), SMCC, Sulfo-SMCC,vinylsilane, N,N′diallyltartardiamide (DATD),N,N′-Bis(acryloyl)cystamine (BAC), or homologs thereof. In some cases,the crosslinker used in the present disclosure contains cystamine.

Crosslinking may be permanent or reversible, depending upon theparticular crosslinker used. Reversible crosslinking may allow for thepolymer to linearize or dissociate under appropriate conditions. In somecases, reversible cross-linking may also allow for reversible attachmentof a material bound to the surface of a bead. In some cases, across-linker may form disulfide linkages. In some cases, the chemicalcross-linker forming disulfide linkages may be cystamine or a modifiedcystamine.

In some embodiments, disulfide linkages can be formed between molecularprecursor units (e.g., monomers, oligomers, or linear polymers) orprecursors incorporated into a bead and oligonucleotides. Cystamine(including modified cystamines), for example, is an organic agentcomprising a disulfide bond that may be used as a crosslinker agentbetween individual monomeric or polymeric precursors of a bead.Polyacrylamide may be polymerized in the presence of cystamine or aspecies comprising cystamine (e.g., a modified cystamine) to generatepolyacrylamide gel beads comprising disulfide linkages (e.g., chemicallydegradable beads comprising chemically-reducible cross-linkers). Thedisulfide linkages may permit the bead to be degraded (or dissolved)upon exposure of the bead to a reducing agent.

In some embodiments, chitosan, a linear polysaccharide polymer, may becrosslinked with glutaraldehyde via hydrophilic chains to form a bead.Crosslinking of chitosan polymers may be achieved by chemical reactionsthat are initiated by heat, pressure, change in pH, and/or radiation.

In some embodiments, the bead may comprise covalent or ionic bondsbetween polymeric precursors (e.g., monomers, oligomers, linearpolymers), oligonucleotides, primers, and other entities. In some cases,the covalent bonds comprise carbon-carbon bonds or thioether bonds.

In some cases, a bead may comprise an acrydite moiety, which in certainaspects may be used to attach one or more oligonucleotides (e.g.,barcode sequence, barcoded oligonucleotide, primer, or otheroligonucleotide) to the bead. In some cases, an acrydite moiety canrefer to an acrydite analogue generated from the reaction of acryditewith one or more species, such as, the reaction of acrydite with othermonomers and cross-linkers during a polymerization reaction. Acryditemoieties may be modified to form chemical bonds with a species to beattached, such as an oligonucleotide (e.g., barcode sequence, barcodedoligonucleotide, primer, or other oligonucleotide). Acrydite moietiesmay be modified with thiol groups capable of forming a disulfide bond ormay be modified with groups already comprising a disulfide bond. Thethiol or disulfide (via disulfide exchange) may be used as an anchorpoint for a species to be attached or another part of the acryditemoiety may be used for attachment. In some cases, attachment isreversible, such that when the disulfide bond is broken (e.g., in thepresence of a reducing agent), the attached species is released from thebead. In other cases, an acrydite moiety comprises a reactive hydroxylgroup that may be used for attachment.

Functionalization of beads for attachment of oligonucleotides may beachieved through a wide range of different approaches, includingactivation of chemical groups within a polymer, incorporation of activeor activatable functional groups in the polymer structure, or attachmentat the pre-polymer or monomer stage in bead production.

For example, precursors (e.g., monomers, cross-linkers) that arepolymerized to form a bead may comprise acrydite moieties, such thatwhen a bead is generated, the bead also comprises acrydite moieties. Theacrydite moieties can be attached to an oligonucleotide, such as aprimer (e.g., a primer for amplifying target nucleic acids, barcodedoligonucleotide, etc) to be incorporated into the bead. In some cases,the primer comprises a P5 sequence for attachment to a sequencing flowcell for Illumina sequencing. In some cases, the primer comprises a P7sequence for attachment to a sequencing flow cell for Illuminasequencing. In some cases, the primer comprises a barcode sequence. Insome cases, the primer further comprises a unique molecular identifier(UMI). In some cases, the primer comprises an R1 primer sequence forIllumina sequencing. In some cases, the primer comprises an R2 primersequence for Illumina sequencing.

In some cases, precursors comprising a functional group that is reactiveor capable of being activated such that it becomes reactive can bepolymerized with other precursors to generate gel beads comprising theactivated or activatable functional group. The functional group may thenbe used to attach additional species (e.g., disulfide linkers, primers,other oligonucleotides, etc.) to the gel beads. For example, someprecursors comprising a carboxylic acid (COOH) group can co-polymerizewith other precursors to form a gel bead that also comprises a COOHfunctional group. In some cases, acrylic acid (a species comprising freeCOOH groups), acrylamide, and bis(acryloyl)cystamine can beco-polymerized together to generate a gel bead comprising free COOHgroups. The COOH groups of the gel bead can be activated (e.g., via1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-Hydroxysuccinimide (NHS) or4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMTMM)) such that they are reactive (e.g., reactive to amine functionalgroups where EDC/NHS or DMTMM are used for activation). The activatedCOOH groups can then react with an appropriate species (e.g., a speciescomprising an amine functional group where the carboxylic acid groupsare activated to be reactive with an amine functional group) comprisinga moiety to be linked to the bead.

Beads comprising disulfide linkages in their polymeric network may befunctionalized with additional species via reduction of some of thedisulfide linkages to free thiols. The disulfide linkages may be reducedvia, for example, the action of a reducing agent (e.g., DTT, TCEP, etc.)to generate free thiol groups, without dissolution of the bead. Freethiols of the beads can then react with free thiols of a species or aspecies comprising another disulfide bond (e.g., via thiol-disulfideexchange) such that the species can be linked to the beads (e.g., via agenerated disulfide bond). In some cases, free thiols of the beads mayreact with any other suitable group. For example, free thiols of thebeads may react with species comprising an acrydite moiety. The freethiol groups of the beads can react with the acrydite via Michaeladdition chemistry, such that the species comprising the acrydite islinked to the bead. In some cases, uncontrolled reactions can beprevented by inclusion of a thiol capping agent such asN-ethylmalieamide or iodoacetate.

Activation of disulfide linkages within a bead can be controlled suchthat only a small number of disulfide linkages are activated. Controlmay be exerted, for example, by controlling the concentration of areducing agent used to generate free thiol groups and/or concentrationof reagents used to form disulfide bonds in bead polymerization. In somecases, a low concentration (e.g., molecules of reducing agent:gel beadratios of less than or equal about 10000, 100000, 1000000, 10000000,100000000, 1000000000, 10000000000, or 100000000000) of reducing agentmay be used for reduction. Controlling the number of disulfide linkagesthat are reduced to free thiols may be useful in ensuring beadstructural integrity during functionalization. In some cases,optically-active agents, such as fluorescent dyes may be may be coupledto beads via free thiol groups of the beads and used to quantify thenumber of free thiols present in a bead and/or track a bead.

In some cases, addition of moieties to a gel bead after gel beadformation may be advantageous. For example, addition of anoligonucleotide (e.g., barcoded oligonucleotide) after gel beadformation may avoid loss of the species during chain transfertermination that can occur during polymerization. Moreover, smallerprecursors (e.g., monomers or cross linkers that do not comprise sidechain groups and linked moieties) may be used for polymerization and canbe minimally hindered from growing chain ends due to viscous effects. Insome cases, functionalization after gel bead synthesis can minimizeexposure of species (e.g., oligonucleotides) to be loaded withpotentially damaging agents (e.g., free radicals) and/or chemicalenvironments. In some cases, the generated gel may possess an uppercritical solution temperature (UCST) that can permit temperature drivenswelling and collapse of a bead. Such functionality may aid inoligonucleotide (e.g., a primer) infiltration into the bead duringsubsequent functionalization of the bead with the oligonucleotide.Post-production functionalization may also be useful in controllingloading ratios of species in beads, such that, for example, thevariability in loading ratio is minimized. Species loading may also beperformed in a batch process such that a plurality of beads can befunctionalized with the species in a single batch.

In some cases, an acrydite moiety linked to precursor, another specieslinked to a precursor, or a precursor itself comprises a labile bond,such as chemically, thermally, or photo-sensitive bonds e.g., disulfidebonds, UV sensitive bonds, or the like. Once acrydite moieties or othermoieties comprising a labile bond are incorporated into a bead, the beadmay also comprise the labile bond. The labile bond may be, for example,useful in reversibly linking (e.g., covalently linking) species (e.g.,barcodes, primers, etc.) to a bead. In some cases, a thermally labilebond may include a nucleic acid hybridization based attachment, e.g.,where an oligonucleotide is hybridized to a complementary sequence thatis attached to the bead, such that thermal melting of the hybridreleases the oligonucleotide, e.g., a barcode containing sequence, fromthe bead or microcapsule.

The addition of multiple types of labile bonds to a gel bead may resultin the generation of a bead capable of responding to varied stimuli.Each type of labile bond may be sensitive to an associated stimulus(e.g., chemical stimulus, light, temperature, etc.) such that release ofspecies attached to a bead via each labile bond may be controlled by theapplication of the appropriate stimulus. Such functionality may beuseful in controlled release of species from a gel bead. In some cases,another species comprising a labile bond may be linked to a gel beadafter gel bead formation via, for example, an activated functional groupof the gel bead as described above. As will be appreciated, barcodesthat are releasably, cleavably or reversibly attached to the beadsdescribed herein include barcodes that are released or releasablethrough cleavage of a linkage between the barcode molecule and the bead,or that are released through degradation of the underlying bead itself,allowing the barcodes to be accessed or accessible by other reagents, orboth.

Species (e.g., oligonucleotides comprising barcodes) attached to a solidsupport (e.g., a bead) may comprise a U-excising element that allows thespecies to release from the bead. In some cases, the U-excising elementmay comprise a single-stranded DNA (ssDNA) sequence that contains atleast one uracil. The species may be attached to a solid support via thessDNA sequence. The species may be released by a combination ofuracil-DNA glycosylase (e.g., to remove the uracil) and an endonuclease(e.g., to induce an ssDNA break). If the endonuclease generates a 5′phosphate group from the cleavage, then additional enzyme treatment maybe included in downstream processing to eliminate the phosphate group,e.g., prior to ligation of additional sequencing handle elements, e.g.,Illumina full P5 sequence, partial P5 sequence, full R1 sequence, and/orpartial sequence.

The barcodes that are releasable as described herein may sometimes bereferred to as being activatable, in that they are available forreaction once released. Thus, for example, an activatable barcode may beactivated by releasing the barcode from a bead (or other suitable typeof partition described herein). Other activatable configurations arealso envisioned in the context of the described methods and systems.

In addition to thermally cleavable bonds, disulfide bonds and UVsensitive bonds, other non-limiting examples of labile bonds that may becoupled to a precursor or bead include an ester linkage (e.g., cleavablewith an acid, a base, or hydroxylamine), a vicinal diol linkage (e.g.,cleavable via sodium periodate), a Diels-Alder linkage (e.g., cleavablevia heat), a sulfone linkage (e.g., cleavable via a base), a silyl etherlinkage (e.g., cleavable via an acid), a glycosidic linkage (e.g.,cleavable via an amylase), a peptide linkage (e.g., cleavable via aprotease), or a phosphodiester linkage (e.g., cleavable via a nuclease(e.g., DNAase)).

Species that do not participate in polymerization may also beencapsulated in beads during bead generation (e.g., duringpolymerization of precursors). Such species may be entered intopolymerization reaction mixtures such that generated beads comprise thespecies upon bead formation. In some cases, such species may be added tothe gel beads after formation. Such species may include, for example,oligonucleotides (e.g. barcoded oligonucleotides and/or anchoroligonucleotides), reagents for a nucleic acid amplification reaction(e.g., primers, polymerases, dNTPs, co-factors (e.g., ionic co-factors))including those described herein, reagents for enzymatic reactions(e.g., enzymes, co-factors, substrates), or reagents for a nucleic acidmodification reactions such as polymerization, ligation, or digestion.Trapping of such species may be controlled by the polymer networkdensity generated during polymerization of precursors, control of ioniccharge within the gel bead (e.g., via ionic species linked topolymerized species), or by the release of other species. Encapsulatedspecies may be released from a bead upon bead degradation and/or byapplication of a stimulus capable of releasing the species from thebead.

Beads may be of uniform size or heterogeneous size. In some cases, thediameter of a bead may be about 1 micrometer (μm), 5 μm, 10 μm, 20 μm,30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm,or 1 mm. In some cases, a bead may have a diameter of at least about 5μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100μm, 250 μm, 500 μm, 1 mm, or more. In some cases, a bead may have adiameter of less than or equal to about 5 μm, 10 μm, 20 μm, 30 μm, 40μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 250 μm, 500 μm, or 1 mm.In some cases, a bead may have a diameter in the range of about 40-75μm, 30-75 μm, 20-75 μm, 40-85 μm, 40-95 μm, 20-100 μm, 10-100 μm, 1-100μm, 20-250 μm, or 20-500 μm.

In certain aspects, beads are provided as a population or plurality ofbeads having a relatively monodisperse size distribution. Suchmonodispersity can provide relatively consistent amounts of reagentswithin partitions and maintain relatively consistent beadcharacteristics. In particular, the beads described herein may have sizedistributions that have a coefficient of variation in theircross-sectional dimensions of less than or equal to about 50%, less thanor equal to about 40%, less than or equal to about 30%, less than orequal to about 20%, less than or equal to about 15%, less than or equalto about 10%, or less than or equal to about 5%.

Beads may be of any suitable shape. Examples of bead shapes include, butare not limited to, spherical, non-spherical, oval, oblong, amorphous,circular, cylindrical, and variations thereof.

In addition to, or as an alternative to the cleavable linkages betweenthe beads and the associated molecules, e.g., barcode containingoligonucleotides, described above, the beads may be degradable,disruptable, or dissolvable spontaneously or upon exposure to one ormore stimuli (e.g., temperature changes, pH changes, exposure toparticular chemical species or phase, exposure to light, reducing agent,etc.). In some cases, a bead may be dissolvable, such that materialcomponents of the beads are solubilized when exposed to a particularchemical species or an environmental change, such as a changetemperature or a change in pH. In some cases, a gel bead is degraded ordissolved at elevated temperature and/or in basic conditions. In somecases, a bead may be thermally degradable such that when the bead isexposed to an appropriate change in temperature (e.g., heat), the beaddegrades. Degradation or dissolution of a bead bound to a species (e.g.,an oligonucleotide, e.g., barcoded oligonucleotide) may result inrelease of the species from the bead.

A degradable bead may comprise one or more species with a labile bondsuch that, when the bead/species is exposed to the appropriate stimuli,the bond is broken and the bead degrades. The labile bond may be achemical bond (e.g., covalent bond, ionic bond) or may be another typeof physical interaction (e.g., van der Waals interactions, dipole-dipoleinteractions, etc.). In some cases, a crosslinker used to generate abead may comprise a labile bond. Upon exposure to the appropriateconditions, the labile bond can be broken and the bead degraded. Forexample, upon exposure of a polyacrylamide gel bead comprising cystaminecrosslinkers to a reducing agent, the disulfide bonds of the cystaminecan be broken and the bead degraded.

A degradable bead may be useful in more quickly releasing an attachedspecies (e.g., an oligonucleotide, a barcode sequence, a primer, etc)from the bead when the appropriate stimulus is applied to the bead ascompared to a bead that does not degrade. For example, for a speciesbound to an inner surface of a porous bead or in the case of anencapsulated species, the species may have greater mobility andaccessibility to other species in solution upon degradation of the bead.In some cases, a species may also be attached to a degradable bead via adegradable linker (e.g., disulfide linker). The degradable linker mayrespond to the same stimuli as the degradable bead or the two degradablespecies may respond to different stimuli. For example, a barcodesequence may be attached, via a disulfide bond, to a polyacrylamide beadcomprising cystamine. Upon exposure of the barcoded-bead to a reducingagent, the bead degrades and the barcode sequence is released uponbreakage of both the disulfide linkage between the barcode sequence andthe bead and the disulfide linkages of the cystamine in the bead.

A degradable bead may be introduced into a partition, such as a dropletof an emulsion or a well, such that the bead degrades within thepartition and any associated species (e.g., oligonucleotides) arereleased within the droplet when the appropriate stimulus is applied.The free species (e.g., oligonucleotides) may interact with otherreagents contained in the partition. For example, a polyacrylamide beadcomprising cystamine and linked, via a disulfide bond, to a barcodesequence, may be combined with a reducing agent within a droplet of awater-in-oil emulsion. Within the droplet, the reducing agent may breakthe various disulfide bonds resulting in bead degradation and release ofthe barcode sequence into the aqueous, inner environment of the droplet.In another example, heating of a droplet comprising a bead-bound barcodesequence in basic solution may also result in bead degradation andrelease of the attached barcode sequence into the aqueous, innerenvironment of the droplet.

As will be appreciated from the above disclosure, while referred to asdegradation of a bead, degradation may refer to the disassociation of abound or entrained species from a bead, both with and withoutstructurally degrading the physical bead itself. For example, entrainedspecies may be released from beads through osmotic pressure differencesdue to, for example, changing chemical environments. By way of example,alteration of bead pore sizes due to osmotic pressure differences cangenerally occur without structural degradation of the bead itself. Insome cases, an increase in pore size due to osmotic swelling of a beadcan permit the release of entrained species within the bead. In othercases, osmotic shrinking of a bead may cause a bead to better retain anentrained species due to pore size contraction.

Where degradable beads are provided, it can be useful to avoid exposingsuch beads to the stimulus or stimuli that cause such degradation priorto a given time, in order to avoid premature bead degradation and issuesthat arise from such degradation, including, for example poor flowcharacteristics and aggregation. By way of example, where beads comprisereducible cross-linking groups, such as disulfide groups, it can beuseful to avoid contacting such beads with reducing agents, e.g., DTT orother disulfide cleaving reagents. In such cases, treatment to the beadsdescribed herein will, in some cases be provided free of reducingagents, such as DTT. Because reducing agents are often provided incommercial enzyme preparations, reducing agent free (or DTT free) enzymepreparations may be provided in treating the beads described herein.Examples of such enzymes include, e.g., polymerase enzyme preparations,reverse transcriptase enzyme preparations, ligase enzyme preparations,as well as many other enzyme preparations that may be used to treat thebeads described herein. The terms “reducing agent free” or “DTT free”preparations can refer to a preparation having less than or equal toabout 1/10th, less than or equal to about 1/50th, or less than or equalto about 1/100th of the lower ranges for such materials used indegrading the beads. For example, for DTT, the reducing agent freepreparation will typically have less than or equal to about 0.01 mM,0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or 0.0001 mM DTT. In some cases,the amount of DTT will be undetectable.

In some cases, a stimulus may be used to trigger degradation of thebead, which may result in the release of contents from the bead.Generally, a stimulus may cause degradation of the bead structure, suchas degradation of the covalent bonds or other types of physicalinteraction. These stimuli may be useful in inducing a bead to degradeand/or to release its contents. Examples of stimuli that may be usedinclude chemical stimuli, thermal stimuli, optical stimuli (e.g., light)and any combination thereof, as described more fully below.

Numerous chemical triggers may be used to trigger the degradation ofbeads. Examples of these chemical changes may include, but are notlimited to pH-mediated changes to the integrity of a component withinthe bead, degradation of a component of a bead via cleavage ofcross-linked bonds, and depolymerization of a component of a bead.

In some embodiments, a bead may be formed from materials that comprisedegradable chemical crosslinkers, such as BAC or cystamine. Degradationof such degradable crosslinkers may be accomplished through a number ofmechanisms. In some examples, a bead may be contacted with a chemicaldegrading agent that may induce oxidation, reduction or other chemicalchanges. For example, a chemical degrading agent may be a reducingagent, such as dithiothreitol (DTT). Additional examples of reducingagents may include β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane(dithiobutylamine or DTBA), tris(2-carboxyethyl) phosphine (TCEP), orcombinations thereof. A reducing agent may degrade the disulfide bondsformed between gel precursors forming the bead, and thus, degrade thebead. In other cases, a change in pH of a solution, such as an increasein pH, may trigger degradation of a bead. In other cases, exposure to anaqueous solution, such as water, may trigger hydrolytic degradation, andthus degradation of the bead.

Beads may also be induced to release their contents upon the applicationof a thermal stimulus. A change in temperature can cause a variety ofchanges to a bead. For example, heat can cause a solid bead to liquefy.A change in heat may cause melting of a bead such that a portion of thebead degrades. In other cases, heat may increase the internal pressureof the bead components such that the bead ruptures or explodes. Heat mayalso act upon heat-sensitive polymers used as materials to constructbeads.

The methods, compositions, devices, and kits of this disclosure may beused with any suitable agent to degrade beads. In some embodiments,changes in temperature or pH may be used to degrade thermo-sensitive orpH-sensitive bonds within beads. In some embodiments, chemical degradingagents may be used to degrade chemical bonds within beads by oxidation,reduction or other chemical changes. For example, a chemical degradingagent may be a reducing agent, such as DTT, wherein DTT may degrade thedisulfide bonds formed between a crosslinker and gel precursors, thusdegrading the bead. In some embodiments, a reducing agent may be addedto degrade the bead, which may or may not cause the bead to release itscontents. Examples of reducing agents may include dithiothreitol (DTT),β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamineor DTBA), tris(2-carboxyethyl) phosphine (TCEP), or combinationsthereof. The reducing agent may be present at a concentration of about0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM. The reducing agent may be presentat a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM,or greater. The reducing agent may be present at concentration of atmost about 0.1 mM, 0.5 mM, 1 mM, 5 mM, or 10 mM.

Any suitable number of nucleic acid molecules (e.g., primer, barcodedoligonucleotide, anchor oligonucleotide) can be associated with a beadsuch that, upon release from the bead, the nucleic acid molecules (e.g.,primer, barcoded oligonucleotide, anchor oligonucleotide) are present inthe partition at a pre-defined concentration. Such pre-definedconcentration may be selected to facilitate certain reactions forgenerating a sequencing library, e.g., amplification, within thepartition. In some cases, the pre-defined concentration of the primer islimited by the process of producing oligonucleotide bearing beads.

In some aspects, the partitions refer to containers or vessels (such aswells, microwells, tubes, vials, through ports in nanoarray substrates,e.g., BioTrove nanoarrays, or other containers). In some aspects, thecompartments or partitions comprise partitions that are flowable withinfluid streams. These partitions may comprise, e.g., micro-vesicles thathave an outer barrier surrounding an inner fluid center or core, or, insome cases, they may comprise a porous matrix that is capable ofentraining and/or retaining materials within its matrix. In someaspects, partitions comprise droplets of aqueous fluid within anon-aqueous continuous phase, e.g., an oil phase. Examples of differentvessels are described in U.S. Patent Application Publication No.2014/0155295, which is entirely incorporated herein by reference for allpurposes. Examples of emulsion systems for creating stable droplets innon-aqueous or oil continuous phases are described in detail in U.S.Patent Application Publication No. 2010/0105112, which is entirelyincorporated herein by reference for all purposes.

In the case of droplets in an emulsion, allocating individual cells todiscrete partitions may generally be accomplished by introducing aflowing stream of cells in an aqueous fluid into a flowing stream of anon-aqueous fluid, such that droplets are generated at the junction ofthe two streams. By providing the aqueous cell-containing stream at acertain concentration of cells, the occupancy of the resultingpartitions (e.g., number of cells per partition) can be controlled.Where single cell partitions are implemented, the relative flow rates ofthe fluids can be selected such that, on average, the partitions containless than one cell per partition, in order to ensure that thosepartitions that are occupied, are primarily singly occupied. In someembodiments, the relative flow rates of the fluids can be selected suchthat a majority of partitions are occupied, e.g., allowing for only asmall percentage of unoccupied partitions. In some aspects, the flowsand channel architectures are controlled as to ensure a number of singlyoccupied partitions, less than a certain level of unoccupied partitionsand less than a certain level of multiply occupied partitions.

The systems and methods described herein can be operated such that amajority of occupied partitions include no more than one cell peroccupied partition. In some cases, the partitioning process is conductedsuch that fewer than 25% of the occupied partitions contain more thanone cell, and in some cases, fewer than 20% of the occupied partitionshave more than one cell. In some cases, fewer than 10% or fewer than 5%of the occupied partitions include more than one cell per partition.

In some cases, it can be useful to avoid the creation of excessivenumbers of empty partitions. For example, from a cost perspective and/orefficiency perspective, it may helpful to minimize the number of emptypartitions. While this may be accomplished by providing sufficientnumbers of cells into the partitioning zone, the Poissonian distributionmay expectedly increase the number of partitions that may includemultiple cells. As such, in accordance with aspects described herein,the flow of one or more of the cells, or other fluids directed into thepartitioning zone are conducted such that, in some cases, no more than50% of the generated partitions, no more than 25% of the generatedpartitions, or no more than 10% of the generated partitions areunoccupied. Further, in some aspects, these flows are controlled so asto present non-Poissonian distribution of single occupied partitionswhile providing lower levels of unoccupied partitions. The above rangesof unoccupied partitions can be achieved while still providing any ofthe single occupancy rates described above. For example, the use of thesystems and methods described herein creates resulting partitions thathave multiple occupancy rates of less than or equal to about 25%, 20%,15%, 10%, or 5%, while having unoccupied partitions of less than orequal to about 50%, 40%, 30%, 20%, 10%, or 5%.

As will be appreciated, the above-described occupancy rates are alsoapplicable to partitions that include both cells and additional reagentsand agents, including, but not limited to, microcapsules carryingbarcoded oligonucleotides, microcapsules carrying anchoringoligonucleotides, labelling agents, labelling agents comprising reporteroligonucleotides, labelling agents comprising reporter oligonucleotidescomprising a nucleic barcode sequence, and cells with one or morelabelling agents bound to one or more cell surface features. In someaspects, a substantial percentage of the overall occupied partitions caninclude a microcapsule (e.g., bead) comprising barcodes or anchoringoligonucleotides and a cell with or without bound labelling agents.

Although described in terms of providing substantially singly occupiedpartitions, above, in certain cases, it can be useful to providemultiply occupied partitions, e.g., containing two, three, four or morecells and/or microcapsules (e.g., beads) comprising barcodedoligonucleotides or anchor oligonucleotides within a single partition.Accordingly, the flow characteristics of the cell and/or bead containingfluids and partitioning fluids may be controlled to provide for suchmultiply occupied partitions. In particular, the flow parameters may becontrolled to provide an occupancy rate at greater than or equal toabout 50% of the partitions, greater than or equal to about 75%, orgreater than or equal to about 80%, 90%, 95%, or higher.

In some cases, additional microcapsules are used to deliver additionalreagents to a partition. In such cases, it may be advantageous tointroduce different beads into a common channel or droplet generationjunction, from different bead sources, i.e., containing differentassociated reagents, through different channel inlets into such commonchannel or droplet generation junction. In such cases, the flow andfrequency of the different beads into the channel or junction may becontrolled to provide for a suitable ratio of microcapsules from eachsource, while ensuring the pairing or combination of such beads into apartition with the number of cells.

The partitions described herein may comprise small volumes, e.g., lessthan or equal to 10 μL, 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL,600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20 pL, 10 pL, 1pL, 500 nanoliters (nL), 100 nL, 50 nL, or less.

For example, in the case of droplet based partitions, the droplets mayhave overall volumes that are less than or equal to 1000 pL, 900 pL, 800pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pL, 100 pL, 50 pL, 20pL, 10 pL, or 1 pL. Where co-partitioned with microcapsules, it will beappreciated that the sample fluid volume, e.g., including co-partitionedcells, within the partitions may be less than or equal to 90%, 80%, 70%,60%, 50%, 40%, 30%, 20%, 10%, or less than the above described volumes.

As is described elsewhere herein, partitioning species may generate apopulation or plurality of partitions. In such cases, any suitablenumber of partitions can be generated to generate the plurality ofpartitions. For example, in a method described herein, a plurality ofpartitions may be generated that comprises at least about 1,000partitions, at least about 5,000 partitions, at least about 10,000partitions, at least about 50,000 partitions, at least about 100,000partitions, at least about 500,000 partitions, at least about 1,000,000partitions, at least about 5,000,000 partitions at least about10,000,000 partitions, at least about 50,000,000 partitions, at leastabout 100,000,000 partitions, at least about 500,000,000 partitions orat least about 1,000,000,000 partitions. Moreover, the plurality ofpartitions may comprise both unoccupied partitions (e.g., emptypartitions) and occupied partitions

Microfluidic channel networks can be utilized to generate partitions asdescribed herein. Alternative mechanisms may also be employed in thepartitioning of individual cells, including porous membranes throughwhich aqueous mixtures of cells are extruded into non-aqueous fluids.

An example of a simplified microfluidic channel structure forpartitioning individual cells is illustrated in FIG. 1. Cells may bepartitioned with or without labelling agents bound to cell surfacefeatures, as described herein. As described herein, in some cases, themajority of occupied partitions include no more than one cell peroccupied partition and, in some cases, some of the generated partitionsare unoccupied. In some cases, though, some of the occupied partitionsmay include more than one cell. In some cases, the partitioning processmay be controlled such that fewer than 25% of the occupied partitionscontain more than one cell, and in some cases, fewer than 20% of theoccupied partitions have more than one cell, while in some cases, fewerthan 10% or fewer than 5% of the occupied partitions include more thanone cell per partition. As shown, the channel structure can includechannel segments 102, 104, 106 and 108 communicating at a channeljunction 110. In operation, a first aqueous fluid 112 that includessuspended cells 114, may be transported along channel segment 102 intojunction 110, while a second fluid 116 that is immiscible with theaqueous fluid 112 is delivered to the junction 110 from channel segments104 and 106 to create discrete droplets 118 of the aqueous fluidincluding individual cells 114, flowing into channel segment 108.

In some aspects, this second fluid 116 comprises an oil, such as afluorinated oil, that includes a fluorosurfactant for stabilizing theresulting droplets, e.g., inhibiting subsequent coalescence of theresulting droplets. Examples of partitioning fluids andfluorosurfactants are described in U.S. Patent Application PublicationNo. 2010/0105112, which is entirely incorporated herein by reference forall purposes.

In other aspects, in addition to or as an alternative to droplet basedpartitioning, cells (with or without labelling agents bound to cellsurface features, as described herein) may be encapsulated within amicrocapsule that comprises an outer shell or layer or porous matrix inwhich is entrained one or more individual cells or small groups ofcells, and may include other reagents. Encapsulation of cells may becarried out by a variety of processes. Such processes combine an aqueousfluid containing the cells to be analyzed with a polymeric precursormaterial that may be capable of being formed into a gel or other solidor semi-solid matrix upon application of a particular stimulus to thepolymer precursor. Such stimuli include, e.g., thermal stimuli (eitherheating or cooling), photo-stimuli (e.g., through photo-curing),chemical stimuli (e.g., through crosslinking, polymerization initiationof the precursor (e.g., through added initiators), or the like.

Preparation of microcapsules comprising cells may be carried out by avariety of methods. For example, air knife droplet or aerosol generatorsmay be used to dispense droplets of precursor fluids into gellingsolutions in order to form microcapsules that include individual cellsor small groups of cells. Likewise, membrane based encapsulation systemsmay be used to generate microcapsules comprising encapsulated cells asdescribed herein. In some aspects, microfluidic systems like that shownin FIG. 1 may be readily used in encapsulating cells as describedherein. In particular, and with reference to FIG. 1, the aqueous fluidcomprising the cells and the polymer precursor material is flowed intochannel junction 110, where it is partitioned into droplets 118comprising the individual cells 114, through the flow of non-aqueousfluid 116. In the case of encapsulation methods, non-aqueous fluid 116may also include an initiator to cause polymerization and/orcrosslinking of the polymer precursor to form the microcapsule thatincludes the entrained cells. Examples of polymer precursor/initiatorpairs are described in U.S. Patent Application Publication No.2014/0378345, which is entirely incorporated herein by reference for allpurposes.

For example, in the case where the polymer precursor material comprisesa linear polymer material, e.g., a linear polyacrylamide, PEG, or otherlinear polymeric material, the activation agent may comprise across-linking agent, or a chemical that activates a cross-linking agentwithin the formed droplets. Likewise, for polymer precursors thatcomprise polymerizable monomers, the activation agent may comprise apolymerization initiator. For example, in certain cases, where thepolymer precursor comprises a mixture of acrylamide monomer with aN,N′-bis-(acryloyl)cystamine (BAC) co-monomer, an agent such astetraethylmethylenediamine (TEMED) may be provided within the secondfluid streams in channel segments 104 and 106, which initiates thecopolymerization of the acrylamide and BAC into a cross-linked polymernetwork or, hydrogel.

Upon contact of the second fluid stream 116 with the first fluid stream112 at junction 110 in the formation of droplets, the TEMED may diffusefrom the second fluid 116 into the aqueous first fluid 112 comprisingthe linear polyacrylamide, which will activate the crosslinking of thepolyacrylamide within the droplets, resulting in the formation of thegel, e.g., hydrogel, microcapsules 118, as solid or semi-solid beads orparticles entraining the cells 114. Although described in terms ofpolyacrylamide encapsulation, other ‘activatable’ encapsulationcompositions may also be employed in the context of the methods andcompositions described herein. For example, formation of alginatedroplets followed by exposure to divalent metal ions, e.g., Ca²⁺, can beused as an encapsulation process using the described processes.Likewise, agarose droplets may also be transformed into capsules throughtemperature based gelling, e.g., upon cooling, or the like. In somecases, encapsulated cells can be selectively releasable from themicrocapsule, e.g., through passage of time, or upon application of aparticular stimulus, that degrades the microcapsule sufficiently toallow the cell, or its contents to be released from the microcapsule,e.g., into a partition, such as a droplet. For example, in the case ofthe polyacrylamide polymer described above, degradation of themicrocapsule may be accomplished through the introduction of anappropriate reducing agent, such as DTT or the like, to cleave disulfidebonds that cross link the polymer matrix. See, e.g., U.S. PatentApplication Publication No. 2014/0378345, which is entirely incorporatedherein by reference for all purposes.

Encapsulated cells or cell populations provide certain potentialadvantages of being storable, and more portable than droplet basedpartitioned cells. Furthermore, in some cases, it may cells to beanalyzed can be incubated for a select period of time, in order tocharacterize changes in such cells over time, either in the presence orabsence of different stimuli. In such cases, encapsulation of individualcells may allow for longer incubation than partitioning in emulsiondroplets, although in some cases, droplet partitioned cells may also beincubated for different periods of time, e.g., at least 10 seconds, atleast 30 seconds, at least 1 minute, at least 5 minutes, at least 10minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 5 hours, or at least 10 hours or more. The encapsulation of cellsmay constitute the partitioning of the cells into which other reagentsare co-partitioned. Alternatively, encapsulated cells may be readilydeposited into other partitions, e.g., droplets, as described above.

In accordance with certain aspects, the cells may be partitioned alongwith lysis reagents in order to release the contents of the cells withinthe partition. In such cases, the lysis agents can be contacted with thecell suspension concurrently with, or immediately prior to theintroduction of the cells into the partitioning junction/dropletgeneration zone, e.g., through an additional channel or channelsupstream of channel junction 110. Examples of lysis agents includebioactive reagents, such as lysis enzymes that are used for lysis ofdifferent cell types, e.g., gram positive or negative bacteria, plants,yeast, mammalian, etc., such as lysozymes, achromopeptidase,lysostaphin, labiase, kitalase, lyticase, and a variety of other lysisenzymes available from, e.g., Sigma-Aldrich, Inc. (St Louis, Mo.), aswell as other commercially available lysis enzymes. Other lysis agentsmay additionally or alternatively be co-partitioned with the cells tocause the release of the cell's contents into the partitions. Forexample, in some cases, surfactant based lysis solutions may be used tolyse cells. In some cases, lysis solutions may include non-ionicsurfactants such as, for example, TritonX-100 and Tween 20. In somecases, lysis solutions may include ionic surfactants such as, forexample, sarcosyl and sodium dodecyl sulfate (SDS). Electroporation,thermal, acoustic or mechanical cellular disruption may also be used incertain cases, e.g., non-emulsion based partitioning such asencapsulation of cells that may be in addition to or in place of dropletpartitioning, where any pore size of the encapsulate is sufficientlysmall to retain nucleic acid fragments of a suitable size, followingcellular disruption.

In addition to the lysis agents co-partitioned with the cells describedabove, other reagents can also be co-partitioned with the cells,including, for example, DNase and RNase inactivating agents orinhibitors, such as proteinase K, chelating agents, such as EDTA, andother reagents employed in removing or otherwise reducing negativeactivity or impact of different cell lysate components on subsequentprocessing of nucleic acids. In addition, in the case of encapsulatedcells, the cells may be exposed to an appropriate stimulus to releasethe cells or their contents from a co-partitioned microcapsule. Forexample, in some cases, a chemical stimulus may be co-partitioned alongwith an encapsulated cell to allow for the degradation of themicrocapsule and release of the cell or its contents into the largerpartition. In some cases, this stimulus may be the same as the stimulusdescribed elsewhere herein for release of oligonucleotides from theirrespective microcapsule (e.g., bead). In alternative aspects, this maybe a different and non-overlapping stimulus, in order to allow anencapsulated cell to be released into a partition at a different timefrom the release of oligonucleotides into the same partition.

Additional reagents may also be co-partitioned with the cells, such asendonucleases to fragment the cell's DNA, DNA polymerase enzymes anddNTPs used to amplify the cell's nucleic acid fragments and to attachthe barcode oligonucleotides to the amplified fragments. Additionalreagents may also include reverse transcriptase enzymes, includingenzymes with terminal transferase activity, primers andoligonucleotides, and switch oligonucleotides (also referred to hereinas “switch oligos” or “template switching oligonucleotides”) which canbe used for template switching. In some cases, template switching can beused to increase the length of a cDNA. In some cases, template switchingcan be used to append a predefined nucleic acid sequence to the cDNA. Inone example of template switching, cDNA can be generated from reversetranscription of a template, e.g., cellular mRNA, where a reversetranscriptase with terminal transferase activity can add additionalnucleotides, e.g., polyC, to the cDNA in a template independent manner.Switch oligos can include sequences complementary to the additionalnucleotides, e.g., polyG. The additional nucleotides (e.g., polyC) onthe cDNA can hybridize to the additional nucleotides (e.g., polyG) onthe switch oligo, whereby the switch oligo can be used by the reversetranscriptase as template to further extend the cDNA. Template switchingoligonucleotides may comprise a hybridization region and a templateregion. The hybridization region can comprise any sequence capable ofhybridizing to the target. In some cases, as previously described, thehybridization region comprises a series of G bases to complement theoverhanging C bases at the 3′ end of a cDNA molecule. The series of Gbases may comprise 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G basesor more than 5 G bases. The template sequence can comprise any sequenceto be incorporated into the cDNA. In some cases, the template regioncomprises at least 1 (e.g., at least 2, 3, 4, 5 or more) tag sequencesand/or functional sequences. Switch oligos may comprise deoxyribonucleicacids; ribonucleic acids; modified nucleic acids including2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverted dT, 5-Methyl dC,2′-deoxyInosine, Super T (5-hydroxybutynl-2′-deoxyuridine), Super G(8-aza-7-deazaguanosine), locked nucleic acids (LNAs), unlocked nucleicacids (UNAs, e.g., UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2′Fluoro bases (e.g., Fluoro C, Fluoro U, Fluoro A, and Fluoro G), or anycombination.

In some cases, the length of a switch oligo may be 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 nucleotidesor longer.

In some cases, the length of a switch oligo may be at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides or longer.

In some cases, the length of a switch oligo may be at most 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 or 250nucleotides.

Additional agents may also be co-partitioned with the cells, such as oneor more labelling agents capable of binding to one or more cell surfacefeatures of the cell(s). Cell surface features may comprise a receptor,an antigen, a surface protein, a transmembrane protein, a cluster ofdifferentiation protein, a protein channel, a protein pump, a carrierprotein, a phospholipid, a glycoprotein, a glycolipid, a cell-cellinteraction protein complex, an antigen-presenting complex, a majorhistocompatibility complex, an engineered T-cell receptor, a T-cellreceptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, and an adherens junction. The labelling agents may comprise anantibody, and antibody fragment, a cell surface receptor bindingmolecule, a receptor ligand, a small molecule, a bi-specific antibody, abi-specific T-cell engager, a T-cell receptor engager, a B-cell receptorengager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and aprotein scaffold. The labelling agents may be coupled, through thecoupling approaches as described herein, to a reporter oligonucleotidecomprising a nucleic acid barcode sequence that permits identificationof the labelling agent, as described herein. In some embodiments, thenucleic acid barcode sequence coupled to the labelling agent maycomprise a unique molecular identifier (UMI) sequence segment, asdescribed herein.

A labelling agent may comprise an antigen presenting particle. In somecases, an antigen presenting particle may comprise an antigen on oradjacent to its surface. The antigen presenting particle may bind to oneor more molecules on the surface of a cell in a sample, e.g., throughthe antigen on the antigen presenting particle. In some cases, anantigen presenting particle may be used as a labelling agent for animmune cell, e.g., a T cell or a B cell. Such antigen presentingparticle may bind to a T cell receptor and/or B cell receptor. In somecases, the antigen presenting particle comprise an antigen that isrecognized (e.g., bound) by an immune cell. The antigen presentingparticle may be a cell, e.g., a cancer cell. The antigen presentingparticle may be a pathogen, e.g., a bacterium, a fungus, a microbe or avirus. In certain cases, the antigen presenting particle (e.g., a cellor a virus) may comprise an antigen expression vector that expresses theantigen on the surface of the particle. The antigen expression vectormay comprise a barcode for identifying the nucleic acid or amino acidsequence of the antigen.

An example method for using an antigen presenting particle to analyze acell may comprise one or more of the following operations. A samplecomprising immune cells (e.g., blood or a fraction thereof) are mixedwith a population of antigen presenting particles, and incubated toallow for the immune cells and antigen presenting particles to interact.The immune cells and antigen presenting particles bound to the immunecells are purified using an antibody that selectively binds to theimmune cells. The bound immune cells and antigen presenting particlesare partitioned into droplets with beads (e.g., gel beads). Each of thebeads comprises anchor oligonucleotide comprising a primer for mRNAmolecules, a barcode and a UMI. At least one of the droplets contains animmune cell, an antigen presenting particle, and a gel bead. The immunecell and the antigen presenting particle in the droplet are lysed. ThemRNA molecules from the immune cell and the antigen presenting particleare released. Reverse transcription is performed with the mRNA moleculesand the anchor oligonucleotide from the bead. Thus, the resulting cDNAare tagged with the barcode and UMI from the anchor oligonucleotide. Theresulting cDNA are then sequenced, e.g., to a high depth per cell on asequencer (e.g., an Illumina sequencer). With the sequence reads, V(D)Jregions of the immune cell are assembled and characteristics of theantigen presenting particle are also determined. When the antigenpresenting particles are cancer cells, mutations and/orsingle-nucleotide polymorphisms (SNPs) may be determined with thesequence reads to identify a sub-populations of tumor cells that aretargeted by an immune cell with the corresponding V(D)J sequences. Whenthe antigen presenting particles are viruses, viral genome may beassembled to identify the sub-clone of viruses that are targeted by theimmune cells with the corresponding V(D)J sequences. The method mayyield pairs of V(D)J sequences and antigen-identifying sequences (e.g.,mRNA of tumor cells or the genome of viruses) that are useful indeveloping personalized immunotherapies or vaccines against specificviral strains.

A protein labeled by a labelling agent (e.g., an antibody labeled by abarcode) may be used as a probe in a binding assay. The protein may bean antibody or a cell surface protein, e.g., a cell receptor such as aT-cell receptor and B-cell receptor. The labelling agent may comprise abarcode and/or a UMI. In some cases, another labelling agent comprisingthe same barcode and/or UMI may be used to analyze nucleic acids fromthe same cell as the protein. The nucleic acids and the protein from thesame cell may be identified by the barcode and/or UMI. In some cases,the nucleic acid sequence of the cell surface protein may be determinedusing the labelling agent for analyzing nucleic acids, so that the aminoacid sequence of the cell surface protein may also be determined. Thelabeled protein from the cell may then be used as a probe in a bindingassay against a target molecule (e.g., a protein). For example, in thebinding assay, whether the labeled cell surface protein can bind to thetarget protein may be determined. The label of the cell surface proteinmay be separated from the cell surface protein, e.g., by denaturation.Then the barcode and/or UMI on the label may be sequenced. The sequencesof the barcode and/or UMI may be used to correlate the binding assayresult with the sequence of the cell surface protein. Thus, theinteraction of the protein with the target molecule may be correlatedwith the sequence of the protein. In some cases, the interaction betweenthe protein and the target molecule may be quantified using the UMI.

Once the contents of the cells are released into their respectivepartitions, the nucleic acids contained therein may be further processedwithin the partitions. In accordance with the methods and systemsdescribed herein, the nucleic acid contents of individual cells can beprovided with unique identifiers such that, upon characterization ofthose nucleic acids they may be attributed as having been derived fromthe same cell or cells. The ability to attribute characteristics toindividual cells or groups of cells is provided by the assignment ofunique identifiers specifically to an individual cell or groups ofcells. Unique identifiers, e.g., in the form of nucleic acid barcodescan be assigned or associated with individual cells or populations ofcells, in order to tag or label the cell's components (and as a result,its characteristics) with the unique identifiers. These uniqueidentifiers can then be used to attribute the cell's components andcharacteristics to an individual cell or group of cells. In someaspects, this is carried out by co-partitioning the individual cells orgroups of cells with the unique identifiers. In some aspects, the uniqueidentifiers are provided in the form of oligonucleotides (also referredto herein as anchor oligonucleotides) that comprise nucleic acid barcodesequences that may be attached to or otherwise associated with thenucleic acid contents of individual cells, or to other components of thecells, and particularly to fragments of those nucleic acids. Theoligonucleotides may be partitioned such that as betweenoligonucleotides in a given partition, the nucleic acid barcodesequences contained therein are the same, but as between differentpartitions, the oligonucleotides can, and do have differing barcodesequences, or at least represent a large number of different barcodesequences across all of the partitions in a given analysis. In someaspects, only one nucleic acid barcode sequence can be associated with agiven partition, although in some cases, two or more different barcodesequences may be present.

The nucleic acid barcode sequences can include from 6 to about 20 ormore nucleotides within the sequence of the oligonucleotides. In somecases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at least 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some cases, thelength of a barcode sequence may be at most 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides maybe completely contiguous, i.e., in a single stretch of adjacentnucleotides, or they may be separated into two or more separatesubsequences that are separated by 1 or more nucleotides. In some cases,separated barcode subsequences can be from about 4 to about 16nucleotides in length. In some cases, the barcode subsequence may be 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In somecases, the barcode subsequence may be at least 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16 nucleotides or longer. In some cases, the barcodesubsequence may be at most 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16nucleotides or shorter.

The co-partitioned oligonucleotides can also comprise other functionalsequences useful in the processing of the nucleic acids from theco-partitioned cells. These sequences include, e.g., targeted orrandom/universal amplification primer sequences for amplifying thegenomic DNA from the individual cells within the partitions whileattaching the associated barcode sequences, sequencing primers or primerrecognition sites, hybridization or probing sequences, e.g., foridentification of presence of the sequences or for pulling down barcodednucleic acids, or any of a number of other potential functionalsequences. Other mechanisms of co-partitioning oligonucleotides may alsobe employed, including, e.g., coalescence of two or more droplets, whereone droplet contains oligonucleotides, or microdispensing ofoligonucleotides into partitions, e.g., droplets within microfluidicsystems. Co-partitioning of oligonucleotides and associated barcodes andother functional sequences or labels, along with sample materials asdescribe herein, may be performed, for example, as described in U.S.Patent Application Publication No. 2014/0227684, which is entirelyincorporated herein by reference for all purposes.

Briefly, in one example, microcapsules, such as beads, are provided thateach include large numbers of the above described barcodedoligonucleotides (also referred to herein as anchor oligonucleotides)releasably attached to the beads, where all of the oligonucleotidesattached to a particular bead will include the same nucleic acid barcodesequence, but where a large number of diverse barcode sequences arerepresented across the population of beads used. In some embodiments,hydrogel beads, e.g., comprising polyacrylamide polymer matrices, areused as a solid support and delivery vehicle for the oligonucleotidesinto the partitions, as they are capable of carrying large numbers ofoligonucleotide molecules, and may be configured to release thoseoligonucleotides upon exposure to a particular stimulus, as describedelsewhere herein. In some cases, the population of beads will provide adiverse barcode sequence library that includes at least 1,000 differentbarcode sequences, at least 5,000 different barcode sequences, at least10,000 different barcode sequences, at least at least 50,000 differentbarcode sequences, at least 100,000 different barcode sequences, atleast 1,000,000 different barcode sequences, at least 5,000,000different barcode sequences, or at least 10,000,000 different barcodesequences. Additionally, each bead can be provided with large numbers ofoligonucleotide molecules attached. In particular, the number ofmolecules of oligonucleotides including the barcode sequence on anindividual bead can be at least 1,000 oligonucleotide molecules, atleast 5,000 oligonucleotide molecules, at least 10,000 oligonucleotidemolecules, at least 50,000 oligonucleotide molecules, at least 100,000oligonucleotide molecules, at least 500,000 oligonucleotides, at least1,000,000 oligonucleotide molecules, at least 5,000,000 oligonucleotidemolecules, at least 10,000,000 oligonucleotide molecules, at least50,000,000 oligonucleotide molecules, at least 100,000,000oligonucleotide molecules, and in some cases at least 1 billionoligonucleotide molecules.

Moreover, when the population of beads is partitioned, the resultingpopulation of partitions can also include a diverse barcode library thatincludes at least 1,000 different barcode sequences, at least 5,000different barcode sequences, at least 10,000 different barcodesequences, at least at least 50,000 different barcode sequences, atleast 100,000 different barcode sequences, at least 1,000,000 differentbarcode sequences, at least 5,000,000 different barcode sequences, or atleast 10,000,000 different barcode sequences. Additionally, eachpartition of the population can include at least 1,000 oligonucleotidemolecules, at least 5,000 oligonucleotide molecules, at least 10,000oligonucleotide molecules, at least 50,000 oligonucleotide molecules, atleast 100,000 oligonucleotide molecules, at least 500,000oligonucleotides, at least 1,000,000 oligonucleotide molecules, at least5,000,000 oligonucleotide molecules, at least 10,000,000 oligonucleotidemolecules, at least 50,000,000 oligonucleotide molecules, at least100,000,000 oligonucleotide molecules, and in some cases at least 1billion oligonucleotide molecules.

In some cases, multiple different barcodes can be incorporated within agiven partition, either attached to a single or multiple beads withinthe partition. For example, in some cases, a mixed, but known barcodesequences set may provide greater assurance of identification in thesubsequent processing, e.g., by providing a stronger address orattribution of the barcodes to a given partition, as a duplicate orindependent confirmation of the output from a given partition.

The oligonucleotides may be releasable from the beads upon theapplication of a particular stimulus to the beads. In some cases, thestimulus may be a photo-stimulus, e.g., through cleavage of aphoto-labile linkage that releases the oligonucleotides. In other cases,a thermal stimulus may be used, where elevation of the temperature ofthe beads environment will result in cleavage of a linkage or otherrelease of the oligonucleotides form the beads. In still other cases, achemical stimulus is used that cleaves a linkage of the oligonucleotidesto the beads, or otherwise results in release of the oligonucleotidesfrom the beads. In one case, such compositions include thepolyacrylamide matrices described above for encapsulation of cells, andmay be degraded for release of the attached oligonucleotides throughexposure to a reducing agent, such as DTT. Examples of other systems andmethods are described in U.S. Patent Application Publication No.2014/0155295 and US. Patent Application Publication No. 2014/0378345,each of which is entirely incorporated herein by reference for allpurposes.

In accordance with the methods and systems described herein, the beadsincluding the attached oligonucleotides may be co-partitioned with theindividual cells, such that a single bead and a single cell arecontained within an individual partition. While single cell/single beadoccupancy is one possible state, it will be appreciated that multiplyoccupied partitions (either in terms of cells, beads or both), orunoccupied partitions (either in terms of cells, beads or both) mayoften be present. An example of a microfluidic channel structure forco-partitioning cells and beads comprising barcode oligonucleotides isschematically illustrated in FIG. 2. As described elsewhere herein, insome aspects, a substantial percentage of the overall occupiedpartitions may include both a bead and a cell and, in some cases, someof the partitions that are generated may be unoccupied. In some cases,some of the partitions may have beads and cells that are not partitioned1:1. In some cases, multiply occupied partitions may be provided, e.g.,containing two, three, four or more cells and/or beads within a singlepartition. As shown, channel segments 202, 204, 206, 208 and 210 areprovided in fluid communication at channel junction 212. An aqueousstream comprising the individual cells 214, is flowed through channelsegment 202 toward channel junction 212. As described above, these cellsmay be suspended within an aqueous fluid, or may have beenpre-encapsulated, prior to the partitioning process.

Concurrently, an aqueous stream comprising the barcode carrying beads216, is flowed through channel segment 204 toward channel junction 212.A non-aqueous partitioning fluid 216 is introduced into channel junction212 from each of side channels 206 and 208, and the combined streams areflowed into outlet channel 210. Within channel junction 212, the twocombined aqueous streams from channel segments 202 and 204 are combined,and partitioned into droplets 218, that include co-partitioned cells 214and beads 216. By controlling the flow characteristics of each of thefluids combining at channel junction 212, as well as controlling thegeometry of the channel junction, partitioning can be optimized toachieve a suitable occupancy level of beads, cells or both, within thepartitions 218 that are generated.

In some cases, lysis agents, e.g., cell lysis enzymes, may be introducedinto the partition with the bead stream, e.g., flowing through channelsegment 204, such that the cell may be lysed at or after the time ofpartitioning. In some cases, cell membranes are maintained intact, suchas to allow for the characterization of cell surface markers, asdescribed later herein. Additional reagents may also be added to thepartition in this configuration, such as endonucleases to fragment thecell's DNA, DNA polymerase enzyme and dNTPs used to amplify the cell'snucleic acid fragments and to attach the barcode oligonucleotides to theamplified fragments. A chemical stimulus, such as DTT, may be used torelease the barcodes from their respective beads into the partition. Insuch cases, the chemical stimulus can be provided along with thecell-containing stream in channel segment 202, such that release of thebarcodes only occurs after the two streams have been combined, e.g.,within the partitions 218. Where the cells are encapsulated, however,introduction of a common chemical stimulus, e.g., that both releases theoligonucleotides form their beads, and releases cells from theirmicrocapsules may generally be provided from a separate additional sidechannel (not shown) upstream of or connected to channel junction 212.

A number of other reagents may be co-partitioned along with the cells,beads, lysis agents and chemical stimuli, including, for example,protective reagents, like proteinase K, chelators, nucleic acidextension, replication, transcription or amplification reagents such aspolymerases, reverse transcriptases, transposases which can be used fortransposon based methods (e.g., Nextera), nucleoside triphosphates orNTP analogues, primer sequences and additional cofactors such asdivalent metal ions used in such reactions, ligation reaction reagents,such as ligase enzymes and ligation sequences, dyes, labels, or othertagging reagents.

The channel networks, e.g., as described herein, can be fluidly coupledto appropriate fluidic components. For example, the inlet channelsegments, e.g., channel segments 202, 204, 206 and 208 are fluidlycoupled to appropriate sources of the materials they are to deliver tochannel junction 212. For example, channel segment 202 may be fluidlycoupled to a source of an aqueous suspension of cells 214 to beanalyzed, while channel segment 204 may be fluidly coupled to a sourceof an aqueous suspension of beads 216. Channel segments 206 and 208 maythen be fluidly connected to one or more sources of the non-aqueousfluid. These sources may include any of a variety of different fluidiccomponents, from simple reservoirs defined in or connected to a bodystructure of a microfluidic device, to fluid conduits that deliverfluids from off-device sources, manifolds, or the like. Likewise, theoutlet channel segment 210 may be fluidly coupled to a receiving vesselor conduit for the partitioned cells. Again, this may be a reservoirdefined in the body of a microfluidic device, or it may be a fluidicconduit for delivering the partitioned cells to a subsequent processoperation, instrument or component.

FIG. 8 shows images of individual Jurkat cells co-partitioned along withbarcode oligonucleotide containing beads in aqueous droplets in anaqueous in oil emulsion. As illustrated, individual cells may be readilyco-partitioned with individual beads. As will be appreciated,optimization of individual cell loading may be carried out by a numberof methods, including by providing dilutions of cell populations intothe microfluidic system in order to achieve suitable cell loading perpartition as described elsewhere herein.

In operation, once lysed, the nucleic acid contents of the individualcells are then available for further processing within the partitions,including, e.g., fragmentation, amplification and barcoding, as well asattachment of other functional sequences. Fragmentation may beaccomplished through the co-partitioning of shearing enzymes, such asendonucleases, in order to fragment the nucleic acids into smallerfragments. These endonucleases may include restriction endonucleases,including type II and type IIs restriction endonucleases as well asother nucleic acid cleaving enzymes, such as nicking endonucleases, andthe like. In some cases, fragmentation may not be implemented, and fulllength nucleic acids may be retained within the partitions, or in thecase of encapsulated cells or cell contents, fragmentation may becarried out prior to partitioning, e.g., through enzymatic methods,e.g., those described herein, or through mechanical methods, e.g.,mechanical, acoustic or other shearing.

Once co-partitioned, and the cells are lysed to release their nucleicacids, the oligonucleotides disposed upon the bead may be used tobarcode and amplify fragments of those nucleic acids. Briefly, in oneaspect, the oligonucleotides present on the beads that areco-partitioned with the cells, are released from their beads into thepartition with the cell's nucleic acids. The oligonucleotides caninclude, along with the barcode sequence, a primer sequence at its5′end. This primer sequence may be a random oligonucleotide sequenceintended to randomly prime numerous different regions on the cell'snucleic acids, or it may be a specific primer sequence targeted to primeupstream of a specific targeted region of the cell's genome.

Once released, the primer portion of the oligonucleotide can anneal to acomplementary region of the cell's nucleic acid. Extension reactionreagents, e.g., DNA polymerase, nucleoside triphosphates, co-factors(e.g., Mg²⁺ or Mn²⁺), that may also be co-partitioned with the cells andbeads, then extend the primer sequence using the cell's nucleic acid asa template, to produce a complementary fragment to the strand of thecell's nucleic acid to which the primer annealed, which complementaryfragment includes the oligonucleotide and its associated barcodesequence. Annealing and extension of multiple primers to differentportions of the cell's nucleic acids will result in a large pool ofoverlapping complementary fragments of the nucleic acid, each possessingits own barcode sequence indicative of the partition in which it wascreated. In some cases, these complementary fragments may themselves beused as a template primed by the oligonucleotides present in thepartition to produce a complement of the complement that again, includesthe barcode sequence. In some cases, this replication process isconfigured such that when the first complement is duplicated, itproduces two complementary sequences at or near its termini, to allowformation of a hairpin structure or partial hairpin structure that mayreduce the ability of the molecule to be the basis for producing furtheriterative copies. As described herein, the cell's nucleic acids mayinclude any nucleic acids within the cell including, for example, thecell's DNA, e.g., genomic DNA, RNA, e.g., messenger RNA, and the like.For example, in some cases, the methods and systems described herein areused in characterizing expressed mRNA, including, e.g., the presence andquantification of such mRNA, and may include RNA sequencing processes asthe characterization process. Alternatively or additionally, thereagents partitioned along with the cells may include reagents for theconversion of mRNA into cDNA, e.g., reverse transcriptase enzymes andreagents, to facilitate sequencing processes where DNA sequencing isemployed. In some cases, where the nucleic acids to be characterizedcomprise RNA, e.g., mRNA, schematic illustration of one example of thisis shown in FIG. 3.

As shown, oligonucleotides that include a barcode sequence areco-partitioned in, e.g., a droplet 302 in an emulsion, along with asample nucleic acid 304. The oligonucleotides 308 may be provided on abead 306 that is co-partitioned with the sample nucleic acid 304, whicholigonucleotides are releasable from the bead 306, as shown in panel A.The oligonucleotides 308 may include a barcode sequence 312, in additionto one or more functional sequences, e.g., sequences 310, 314 and 316.For example, oligonucleotide 308 is shown as comprising barcode sequence312, as well as sequence 310 that may function as an attachment orimmobilization sequence for a given sequencing system, e.g., a P5sequence used for attachment in flow cells of an Illumina Hiseq® orMiseq® system. As shown, the oligonucleotides also include a primersequence 316, which may include a random or targeted N-mer for primingreplication of portions of the sample nucleic acid 304. Also includedwithin oligonucleotide 308 is a sequence 314 which may provide asequencing priming region, such as a “read1” or R1 priming region, thatis used to prime polymerase mediated, template directed sequencing bysynthesis reactions in sequencing systems. As will be appreciated, thefunctional sequences may be selected to be compatible with a variety ofdifferent sequencing systems, e.g., 454 Sequencing, Ion Torrent Protonor PGM, Illumina X10, etc., and the requirements thereof. In some cases,the barcode sequence 312, immobilization sequence 310 and R1 sequence314 may be common to all of the oligonucleotides attached to a givenbead. The primer sequence 316 may vary for random N-mer primers, or maybe common to the oligonucleotides on a given bead for certain targetedapplications. Moreover, in some cases, barcoded oligonucleotides may begenerated as described in U.S. Patent Publication No. 20160257984, whichis herein incorporated by reference in its entirety.

An oligonucleotide of an anchor agent or a labelling agent may comprisemodifications that render it non-extendable by a polymerase. Whenbinding to a nucleic acid in a sample for a primer extension reaction,the oligonucleotide may serve as a template, not a primer. When theoligonucleotide also comprises a barcode (e.g., the oligonucleotide is areporter oligonucleotide), such design may increase the efficiency ofmolecular barcoding by increasing the affinity between theoligonucleotide and the unbarcoded sample nucleic acids, and eliminatethe potential formation of adaptor artifacts. In some cases, theoligonucleotide may comprise a random N-mer sequence that is capped withmodifications that render it non-extendable by a polymerase. In somecases, the composition of the random N-mer sequence may be designed tomaximize the binding efficiency to free, unbarcoded ssDNA molecules. Thedesign may include a random sequence composition with a higher GCcontent, a partial random sequence with fixed G or C at specificpositions, the use of guanosines, the use of locked nucleic acids, orany combination thereof.

A modification for blocking primer extension by a polymerase may be acarbon spacer group of different lengths or a dideoxynucleotide. In somecases, the modification may be an abasic site that has an apurine orapyrimidine structure, a base analog, or an analogue of a phosphatebackbone, such as a backbone of N-(2-aminoethyl)-glycine linked by amidebonds, tetrahydrofuran, or 1′,2′-Dideoxyribose. The modification mayalso be a uracil base, 2′OMe modified RNA, C3-18 spacers (e.g.,structures with 3-18 consecutive carbon atoms, such as C3 spacer),ethylene eglycol multimer spacers (e.g., spacer 18 (hexa-ethyleneglycolspacer), biotin, di-deoxynucleotide triphosphate, ethylene glycol,amine, or phosphate.

FIG. 21 shows an oligonucleotide with such modification. Thedouble-stranded oligonucleotide 2110 comprises a single-strandedDNA(ssDNA) annealing region with a random N-mer sequence at its 3′ end.The unbarcoded ssDNA 2120 from a sample binds to oligonucleotide 2110.The random N-mer sequence of the oligonucleotide 2110 has modifications(shown as “X”) on the 3′ end. When oligonucleotide 2110 and unbarcodedssDNA 2120 bind to each other in a primer extension reaction, onlyunbarcoded ssDNA 2120 can be extended using oligonucleotide 3310 as atemplate.

In some cases, the oligonucleotide with a random N-mer sequence may becoupled to a solid support (e.g., a bead) via a U-excising element,e.g., an ssDNA sequence with uracil. FIG. 22 shows an example of sucholigonucleotide. Double-stranded oligonucleotide 2210 comprises an ssDNAannealing region that contains a random N-mer sequence at its 3′ end.Oligonucleotide 2210 is coupled to a bead via an ssDNA 2211 that has auracil. Oligonucleotide 2210 also comprises modifications preventingextension by a polymerase. Oligonucleotide 2210 may be released from thebead by uracil-DNA glycosylase (to remove the uracil) and anendonuclease (to induce the ssDNA break), resulting the releasedoligonucleotide 2230. Oligonucleotide 2220 comprises an ssDNA primingregion has similar design as Oligonucleotide 2210. In some cases, thedifference between an ssDNA annealing region and an ssDNA priming regionis the presence or absence of a blocking group (e.g., “X”),respectively. Unblocked ssDNA can be extended and function as a primer,while blocked ssDNA can function as a passive annealing sequence.

As will be appreciated, in some cases, the functional sequences mayinclude primer sequences useful for RNA-seq applications. For example,in some cases, the oligonucleotides may include poly-T primers forpriming reverse transcription of RNA for RNA-seq. In still other cases,oligonucleotides in a given partition, e.g., included on an individualbead, may include multiple types of primer sequences in addition to thecommon barcode sequences, such as both DNA-sequencing and RNA sequencingprimers, e.g., poly-T primer sequences included within theoligonucleotides coupled to the bead. In such cases, a singlepartitioned cell may be both subjected to DNA and RNA sequencingprocesses.

A primer on a labelling agent or an anchor agent (e.g., a primer forRNA-seq applications) may be a target-specific primer. A target-specificprimer may bind to a specific sequence in a RNA molecule or a DNAmolecule (e.g., complementary DNA (cDNA) from RNA, or endogenous DNAfrom a cell). For example, the specific sequence may be a sequence thatis not in the poly-A tail of an RNA molecule or its cDNA. In some cases,the target-specific primer may bind to RNA molecules such as mRNAmolecules or non-coding RNA molecules, e.g., rRNA, tRNA, mRNA, or miRNAmolecules. In some cases, the target-specific primer may bind to RNAmolecules introduced to a cell. In some cases, the RNA moleculesintroduced to a cell may be RNA molecules used in gene editing methods(e.g., Clustered regularly interspaced short palindromic repeats(CRISPR) RNA (crRNA) or guide RNA for CRISPR gene editing). For example,the target-specific primer may bind to crRNA for identifying the crRNAintroduced to a cell and/or determining the effect of the crRNA on thetranscriptome of the cell. In some cases, the target-specific primer maybe used to determine copy numbers of disease (e.g., cancer)-relatedgenes while simultaneously analyzing the rest of the transcriptome. Inother cases, the target-specific primer may be used to analyze RNAmolecules from pathogens infecting the cell, e.g., for distinguishingpathogen infected cells from non-pathogen infected cells and/ordetermining how the pathogen alters the cells transcriptome. In somecases, a target-specific primer may bind to DNA molecules, e.g.,endogenous DNA molecules from a cell, or synthetic DNA molecules. Forexample, a target-specific primer may bind to a barcode, e.g., a barcodeof a cell (e.g., inside a cell or on the surface of a cell), a barcodeof a protein (e.g., an antibody barcode), or a barcode of a nucleic acid(e.g., a CRISPR barcode).

A target-specific primer may be combined with one or more barcodes, oneor more UMIs, one or more poly-T primers for mRNA, and/or one or morerandom N-mer primers (randomers) for total RNA in the same or differentoligonucleotides. In some cases, a bead disclosed herein may comprise anoligonucleotide with a target-specific primer and one or moreoligonucleotides with a poly-T primer, e.g., as shown in FIG. 23A. Insome cases, a bead may have a plurality of oligonucleotides, each ofwhich comprises a target-specific primer, e.g., as shown in FIG. 23B. Insome cases, a bead may have a plurality of oligonucleotides, each ofwhich comprises a target-specific primer and a plurality ofoligonucleotides, each of which comprises a poly-T primer, e.g., asshown in FIG. 23C. In some cases, a bead may have a plurality ofoligonucleotides, each of which comprises a target-specific primer and aplurality of oligonucleotides, each of which comprises a random N-merprimer for total RNA, e.g., as shown in FIG. 24.

On a bead, the ratio of oligonucleotides with target-specific primers tooligonucleotides with non-specific (poly-T or random N-mer) primers maybe adjusted to match the needs of a specific application. In some cases,at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, or 100% of the oligonucleotides on a bead may comprisetarget-specific primers. In some cases, at least 0.1%, 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of theoligonucleotides on a bead may comprise non-specific (poly-T or randomN-mer) primers. The oligonucleotide may be made by attaching (e.g., byligation) one or more oligonucleotide backbones on a bead and thenattaching (e.g., by ligation) one or more primer sequences to thebackbones.

An oligonucleotide of an anchor agent or a labelling agent may be asplint oligonucleotide. A splint oligonucleotide may comprise two ormore different primers. The primers may have different functions. Forexample, a splint oligonucleotide may comprise two or more of thefollowing: a poly-T primer, a random N-mer primmer, and atarget-specific primer.

An oligonucleotide of an anchor agent or a labelling agent may comprisean adapter that is capable of binding or ligating to an assay primer.The adapter may allow the anchor agent or the labelling agent to beattached to any suitable assay primers and used in any suitable assays.The assay primer may comprise priming region and a sequence that iscapable of binding or ligating to the adapter. In some cases, theadapter may be a non-specific primer (e.g., a 5′ overhang) and the assayprimer may comprise a 3′ overhang that can be ligated to the 5′overhang. The priming region on the assay primer may be any primerdescribed herein, e.g., a poly-T primer, a random N-mer primer, or atarget-specific primer. FIG. 25 shows adapters and assay primers.Oligonucleotide 2510 comprises an adapter 2511, which is a 5′ overhangcomprising 10 nucleotides. The adapter 2511 can be ligated to the assayprimers, each of which comprises a 3′ overhang comprising 10 nucleotidesthat complementary to the 5′ overhang of adapter 2511. The anchoroligonucleotide may be used in any assay by attaching to the assayprimer designed for that assay. In some cases, the adapter comprise aswitch oligo, e.g., with a 3′ end 3rG. FIG. 26 shows an adaptercomprising a switch oligo.

Based upon the presence of primer sequence 316, the oligonucleotides canprime the sample nucleic acid as shown in panel B, which allows forextension of the oligonucleotides 308 and 308 a using polymerase enzymesand other extension reagents also co-partitioned with the bead 306 andsample nucleic acid 304. As shown in panel C, following extension of theoligonucleotides that, for random N-mer primers, may anneal to multipledifferent regions of the sample nucleic acid 304; multiple overlappingcomplements or fragments of the nucleic acid are created, e.g.,fragments 318 and 320. Although including sequence portions that arecomplementary to portions of sample nucleic acid, e.g., sequences 322and 324, these constructs are generally referred to herein as comprisingfragments of the sample nucleic acid 304, having the attached barcodesequences.

The barcoded nucleic acid fragments may then be subjected tocharacterization, e.g., through sequence analysis, or they may befurther amplified in the process, as shown in panel D. For example,additional oligonucleotides, e.g., oligonucleotide 308 b, also releasedfrom bead 306, may prime the fragments 318 and 320. This shown forfragment 318. In particular, again, based upon the presence of therandom N-mer primer 316 b in oligonucleotide 308 b (which in some casescan be different from other random N-mers in a given partition, e.g.,primer sequence 316), the oligonucleotide anneals with the fragment 318,and is extended to create a complement 326 to at least a portion offragment 318 which includes sequence 328, that comprises a duplicate ofa portion of the sample nucleic acid sequence. Extension of theoligonucleotide 308 b continues until it has replicated through theoligonucleotide portion 308 of fragment 318. As illustrated in panel D,the oligonucleotides may be configured to prompt a stop in thereplication by the polymerase at a given point, e.g., after replicatingthrough sequences 316 and 314 of oligonucleotide 308 that is includedwithin fragment 318. As described herein, this may be accomplished bydifferent methods, including, for example, the incorporation ofdifferent nucleotides and/or nucleotide analogues that are not capableof being processed by the polymerase enzyme used. For example, this mayinclude the inclusion of uracil containing nucleotides within thesequence region 312 to prevent a non-uracil tolerant polymerase to ceasereplication of that region. As a result a fragment 326 is created thatincludes the full-length oligonucleotide 308 b at one end, including thebarcode sequence 312, the attachment sequence 310, the R1 primer region314, and the random N-mer sequence 316 b. At the other end of thesequence may be included the complement 316′ to the random N-mer of thefirst oligonucleotide 308, as well as a complement to all or a portionof the R1 sequence, shown as sequence 314′. The R1 sequence 314 and itscomplement 314′ are then able to hybridize together to form a partialhairpin structure 328. As will be appreciated because the random N-mersdiffer among different oligonucleotides, these sequences and theircomplements may not be expected to participate in hairpin formation,e.g., sequence 316′, which is the complement to random N-mer 316, maynot be expected to be complementary to random N-mer sequence 316 b. Thismay not be the case for other applications, e.g., targeted primers,where the N-mers may be common among oligonucleotides within a givenpartition.

By forming these partial hairpin structures, it allows for the removalof first level duplicates of the sample sequence from furtherreplication, e.g., preventing iterative copying of copies. The partialhairpin structure also provides a useful structure for subsequentprocessing of the created fragments, e.g., fragment 326.

In general, the amplification of the cell's nucleic acids is carried outuntil the barcoded overlapping fragments within the partition constituteat least 1× coverage of the particular portion or all of the cell'sgenome, at least 2×, at least 3×, at least 4×, at least 5×, at least10×, at least 20×, at least 40× or more coverage of the genome or itsrelevant portion of interest. Once the barcoded fragments are produced,they may be directly sequenced on an appropriate sequencing system,e.g., an Illumina Hiseq®, Miseq® or X10 system, or they may be subjectedto additional processing, such as further amplification, attachment ofother functional sequences, e.g., second sequencing primers, for reversereads, sample index sequences, and the like.

All of the fragments from multiple different partitions may then bepooled for sequencing on high throughput sequencers as described herein,where the pooled fragments comprise a large number of fragments derivedfrom the nucleic acids of different cells or small cell populations, butwhere the fragments from the nucleic acids of a given cell will sharethe same barcode sequence. In particular, because each fragment is codedas to its partition of origin, and consequently its single cell or smallpopulation of cells, the sequence of that fragment may be attributedback to that cell or those cells based upon the presence of the barcode,which will also aid in applying the various sequence fragments frommultiple partitions to assembly of individual genomes for differentcells. This is schematically illustrated in FIG. 4. As shown in oneexample, a first nucleic acid 404 from a first cell 400, and a secondnucleic acid 406 from a second cell 402 are each partitioned along withtheir own sets of barcode oligonucleotides as described above. Thenucleic acids may comprise a chromosome, entire genome or other largenucleic acid from the cells.

Within each partition, each cell's nucleic acids 404 and 406 is thenprocessed to separately provide overlapping set of second fragments ofthe first fragment(s), e.g., second fragment sets 408 and 410. Thisprocessing also provides the second fragments with a barcode sequencethat is the same for each of the second fragments derived from aparticular first fragment. As shown, the barcode sequence for secondfragment set 408 is denoted by “1” while the barcode sequence forfragment set 410 is denoted by “2”. A diverse library of barcodes may beused to differentially barcode large numbers of different fragment sets.However, it is not necessary for every second fragment set from adifferent first fragment to be barcoded with different barcodesequences. In some cases, multiple different first fragments may beprocessed concurrently to include the same barcode sequence. Diversebarcode libraries are described in detail elsewhere herein.

The barcoded fragments, e.g., from fragment sets 408 and 410, may thenbe pooled for sequencing using, for example, sequence by synthesistechnologies available from Illumina or Ion Torrent division ofThermo-Fisher, Inc. Once sequenced, the sequence reads 412 can beattributed to their respective fragment set, e.g., as shown inaggregated reads 414 and 416, at least in part based upon the includedbarcodes, and in some cases, in part based upon the sequence of thefragment itself. The attributed sequence reads for each fragment set arethen assembled to provide the assembled sequence for each cell's nucleicacids, e.g., sequences 418 and 420, which in turn, may be attributed toindividual cells, e.g., cells 400 and 402.

While described in terms of analyzing the genetic material presentwithin cells, the methods and systems described herein may have muchbroader applicability, including the ability to characterize otheraspects of individual cells or cell populations, by allowing for theallocation of reagents and/or agents to individual cells, and providingfor the attributable analysis or characterization of those cells inresponse to those reagents and/or agents. These methods and systems maybe valuable in being able to characterize cells for, e.g., research,diagnostic, or pathogen identification. By way of example, a wide rangeof different cell surface features, e.g., cell surface proteins likecluster of differentiation or CD proteins, have significant diagnosticrelevance in characterization of diseases like cancer.

In one particularly useful application, the methods and systemsdescribed herein may be used to characterize cell features, such as cellsurface features. Cell surface features may include, but are not limitedto, a receptor, an antigen, a surface protein, a transmembrane protein,a cluster of differentiation protein, a protein channel, a protein pump,a carrier protein, a phospholipid, a glycoprotein, a glycolipid, acell-cell interaction protein complex, an antigen-presenting complex, amajor histocompatibility complex, an engineered T-cell receptor, aT-cell receptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, and an adherens junction. In particular, the methods describedherein may be used to attach one or more labelling agents to these cellfeatures, that when partitioned as described above, may be barcoded andanalyzed, e.g., using DNA sequencing technologies, to ascertain thepresence, and in some cases, relative abundance or quantity of such cellfeatures of an individual cell or population of cells.

In a particular example, a library of potential cell surface featurelabelling agents may be provided associated with a first set of nucleicacid reporter molecules, e.g., where a different reporteroligonucleotide sequence is associated with a specific labelling agent,and therefore capable of binding to a specific cell surface feature.Cell surface feature labelling agents may include, but are not limitedto, an antibody, an antibody fragment, a cell surface receptor bindingmolecule, a receptor ligand, a small molecule, a bi-specific antibody, abi-specific T-cell engager, a T-cell receptor engager, a B-cell receptorengager, a pro-body, an aptamer, a monobody, an affimer, a darpin, and aprotein scaffold. In some aspects, different members of the library maybe characterized by the presence of a different oligonucleotide sequencelabel, e.g., an antibody to a first type of cell surface protein orreceptor may have associated with it a first known reporteroligonucleotide sequence, while an antibody to a second receptor proteinmay have a different known reporter oligonucleotide sequence associatedwith it. Prior to co-partitioning, the cells may be incubated with thelibrary of labelling agents, that may represent antibodies to a broadpanel of different cell surface features, e.g., receptors, proteins,etc., and which include their associated reporter oligonucleotides.Unbound labelling agents may be washed from the cells, and the cells maythen be co-partitioned along with the barcode oligonucleotides describedabove. As a result, the partitions may include the cell or cells, aswell as the bound labelling agents and their known, associated reporteroligonucleotides.

Without the need for lysing the cells within the partitions, one maythen subject the reporter oligonucleotides to the barcoding operationsdescribed above for cellular nucleic acids, to produce barcoded,reporter oligonucleotides, where the presence of the reporteroligonucleotides can be indicative of the presence of the particularcell surface feature, and the barcode sequence will allow theattribution of the range of different cell surface features to a givenindividual cell or population of cells based upon the barcode sequencethat was co-partitioned with that cell or population of cells. As aresult, one may generate a cell-by-cell profile of the cell surfacefeatures within a broader population of cells. This aspect of themethods and systems described herein, is described in greater detailbelow.

This example is schematically illustrated in FIG. 5. As shown, apopulation of cells, represented by cells 502 and 504 are incubated witha library of cell surface associated labelling agents, e.g., antibodies,antibody fragments, cell surface receptor binding molecules, receptorligands, small molecules, bi-specific antibodies, bi-specific T-cellengagers, T-cell receptor engagers, B-cell receptor engagers,pro-bodies, aptamers, monobodies, affimers, darpins, protein scaffolds,or the like, where each different type of binding group includes anassociated nucleic acid reporter molecule associated with it, shown aslabelling agents and associated reporter oligonucleotide 506, 508, 510and 512 (with the reporter oligonucleotides being indicated by thedifferently shaded circles). Where the cell expresses the surfacefeatures that are bound by the library of labelling agents, thelabelling agents and their associated reporter oligonucleotides canbecome associated or coupled with the cell surface feature. Individualcells may then be partitioned into separate partitions, e.g., droplets514 and 516, as described herein, along with their associated labellingagents/reporter oligonucleotides, as well as a bead containingindividual barcode oligonucleotides (e.g., anchor oligonucleotides) asdescribed elsewhere herein, e.g., beads 518 and 520, respectively. Aswith other examples described herein, the barcoded oligonucleotides maybe released from the beads and used to attach the barcode sequence thereporter oligonucleotides present within each partition with a barcodethat is common to a given partition, but which varies widely amongdifferent partitions. For example, as shown in FIG. 5, the reporteroligonucleotides that associate with cell 502 in partition 514 arebarcoded with barcode sequence 522, while the reporter oligonucleotidesassociated with cell 504 in partition 516 are barcoded with barcodesequence 524. As a result, one is provided with a library ofoligonucleotides that reflects the surface features of the cell, asreflected by the reporter molecule, but which is substantiallyattributable to an individual cell by virtue of a common barcodesequence, allowing a single cell level profiling of the surfacecharacteristics of the cell. As will be appreciated, this process is notlimited to cell surface receptors but may be used to identify thepresence of a wide variety of specific cell structures, chemistries orother characteristics.

Single cell processing and analysis methods and systems described hereincan be utilized for a wide variety of applications, including analysisof specific individual cells, analysis of different cell types withinpopulations of differing cell types, analysis and characterization oflarge populations of cells for environmental, human health,epidemiological forensic, or any of a wide variety of differentapplications.

A particularly valuable application of the single cell analysisprocesses described herein is in the sequencing and characterization ofa diseased cell. A diseased cell can have altered metabolic properties,gene expression, protein expression, and/or morphologic features.Examples of diseases include inflammatory disorders, metabolicdisorders, nervous system disorders, and cancer.

Of particular interest are cancer cells. In particular, conventionalanalytical techniques, including the ensemble sequencing processesalluded to above, are not highly adept at picking small variations ingenomic make-up of cancer cells, particularly where those exist in a seaof normal tissue cells. Further, even as between tumor cells, widevariations can exist and can be masked by the ensemble approaches tosequencing (See, e.g., Patel, et al., Single-cell RNA-seq highlightsintratumoral heterogeneity in primary glioblastoma, Science DOI:10.1126/science.1254257 (Published online Jun. 12, 2014). Cancer cellsmay be derived from solid tumors, hematological malignancies, celllines, or obtained as circulating tumor cells, and subjected to thepartitioning processes described above. Upon analysis, one can identifyindividual cell sequences as deriving from a single cell or small groupof cells, and distinguish those over normal tissue cell sequences.

Non-limiting examples of cancer cells include cells of cancers such asAcanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginousmelanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblasticleukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia,Acute myeloblastic leukemia with maturation, Acute myeloid dendriticcell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia,Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma,Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cellleukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers,AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma,Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer,Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma,Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basalcell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma,Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma,Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer,Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Browntumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, CarcinoidTumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinomaof Unknown Primary Site, Carcinosarcoma, Castleman's Disease, CentralNervous System Embryonal Tumor, Cerebellar Astrocytoma, CerebralAstrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma,Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma,Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronicmyelogenous leukemia, Chronic Myeloproliferative Disorder, Chronicneutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectalcancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease,Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small roundcell tumor, Diffuse large B cell lymphoma, Dysembryoplasticneuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor,Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor,Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma,Epithelioid sarcoma, Erythroleukemia, Esophageal cancer,Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma,Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ CellTumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease,Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicularlymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladdercancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma,Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor,Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germcell tumor, Germinoma, Gestational choriocarcinoma, GestationalTrophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme,Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma,Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head andNeck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma,Hemangiopericytoma, Hemangio sarcoma, Hematological malignancy,Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditarybreast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma,Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer,Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenilemyelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, KidneyCancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngealcancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and OralCavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma,Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma,Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibroushistiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma,Malignant Mesothelioma, Malignant peripheral nerve sheath tumor,Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantlecell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor,Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma,Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma,Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic SquamousNeck Cancer with Occult Primary, Metastatic urothelial carcinoma, MixedMullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor,Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiplemyeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease,Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma,Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, NasopharyngealCancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma,Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-HodgkinLymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small CellLung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma,Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer,Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer,Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor,Ovarian Low Malignant Potential Tumor, Paget's disease of the breast,Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroidcancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer,Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor,Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor ofIntermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitaryadenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonaryblastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primarycentral nervous system lymphoma, Primary effusion lymphoma, PrimaryHepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer,Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxomaperitonei, Rectal Cancer, Renal cell carcinoma, Respiratory TractCarcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma,Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygealteratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceousgland carcinoma, Secondary neoplasm, Seminoma, Serous tumor,Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome,Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor,Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Smallintestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart,Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma,Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma,Supratentorial Primitive Neuroectodermal Tumor, Surfaceepithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblasticleukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia,T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminallymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, ThymicCarcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of RenalPelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethralcancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, VaginalCancer, Verner Morrison syndrome, Verrucous carcinoma, Visual PathwayGlioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor,Wilms' tumor, and combinations thereof.

Where cancer cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions may comprise gene specific sequences whichtarget genes or regions of genes associated with or suspected of beingassociated with cancer. For example, this can include genes or regionsof genes where the presence of mutations (e.g., insertions, deletions,polymorphisms, copy number variations, and gene fusions) associated witha cancerous condition are suspected to be present in a cell population.

As with cancer cell analysis, the analysis and diagnosis of fetal healthor abnormality through the analysis of fetal cells is a difficult taskusing conventional techniques. In particular, in the absence ofrelatively invasive procedures, such as amniocentesis obtaining fetalcell samples can employ harvesting those cells from the maternalcirculation. As will be appreciated, such circulating fetal cells makeup an extremely small fraction of the overall cellular population ofthat circulation. As a result complex analyses are performed in order tocharacterize what of the obtained data is likely derived from fetalcells as opposed to maternal cells. By employing the single cellcharacterization methods and systems described herein, however, one canattribute genetic make up to individual cells, and categorize thosecells as maternal or fetal based upon their respective genetic make-up.Further, the genetic sequence of fetal cells may be used to identify anyof a number of genetic disorders, including, e.g., aneuploidy such asDown syndrome, Edwards syndrome, and Patau syndrome. Further, the cellsurface features of fetal cells may be used to identify any of a numberof disorders or diseases.

Also of interest are immune cells. The methods, compositions, andsystems disclosed herein can be utilized for sequence analysis of theimmune repertoire, including genomic, proteomic, and cell surfacefeatures. Analysis of information underlying the immune repertoire canprovide a significant improvement in understanding the status andfunction of the immune system.

Non-limiting examples of immune cells which can be analyzed utilizingthe methods described herein include B cells, T cells (e.g., cytotoxic Tcells, natural killer T cells, regulatory T cells, and T helper cells),natural killer cells, cytokine induced killer (CIK) cells; myeloidcells, such as granulocytes (basophil granulocytes, eosinophilgranulocytes, neutrophil granulocytes/hypersegmented neutrophils),monocytes/macrophages, mast cell, thrombocytes/megakaryocytes, anddendritic cells. In some embodiments, individual T cells are analyzedusing the methods disclosed herein. In some embodiments, individual Bcells are analyzed using the methods disclosed herein.

Immune cells express various adaptive immunological receptors relatingto immune function, such as T cell receptors and B cell receptors. Tcell receptors and B cells receptors play a part in the immune responseby specifically recognizing and binding to antigens and aiding in theirdestruction.

The T cell receptor, or TCR, is a molecule found on the surface of Tcells that is generally responsible for recognizing fragments of antigenas peptides bound to major histocompatibility complex (MHC) molecules.The TCR is generally a heterodimer of two chains, each of which is amember of the immunoglobulin superfamily, possessing an N-terminalvariable (V) domain, and a C terminal constant domain. In humans, in 95%of T cells the TCR consists of an alpha (α) and beta (β) chain, whereasin 5% of T cells the TCR consists of gamma and delta (γ/δ) chains. Thisratio can change during ontogeny and in diseased states as well as indifferent species. When the TCR engages with antigenic peptide and MHC(peptide/MHC), the T lymphocyte is activated through signaltransduction.

Each of the two chains of a TCR contains multiple copies of genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining T gene segment. The TCR alpha chain is generated byrecombination of V and J segments, while the beta chain is generated byrecombination of V, D, and J segments. Similarly, generation of the TCRgamma chain involves recombination of V and J gene segments, whilegeneration of the TCR delta chain occurs by recombination of V, D, and Jgene segments. The intersection of these specific regions (V and J forthe alpha or gamma chain, or V, D and J for the beta or delta chain)corresponds to the CDR3 region that is important for antigen-MHCrecognition. Complementarity determining regions (e.g., CDR1, CDR2, andCDR3), or hypervariable regions, are sequences in the variable domainsof antigen receptors (e.g., T cell receptor and immunoglobulin) that cancomplement an antigen. Most of the diversity of CDRs is found in CDR3,with the diversity being generated by somatic recombination eventsduring the development of T lymphocytes. A unique nucleotide sequencethat arises during the gene arrangement process can be referred to as aclonotype.

The B cell receptor, or BCR, is a molecule found on the surface of Bcells. The antigen binding portion of a BCR is composed of amembrane-bound antibody that, like most antibodies (e.g.,immunoglobulins), has a unique and randomly determined antigen-bindingsite. The antigen binding portion of a BCR includes membrane-boundimmunoglobulin molecule of one isotype (e.g., IgD, IgM, IgA, IgG, orIgE). When a B cell is activated by its first encounter with a cognateantigen, the cell proliferates and differentiates to generate apopulation of antibody-secreting plasma B cells and memory B cells. Thevarious immunoglobulin isotypes differ in their biological features,structure, target specificity and distribution. A variety of molecularmechanisms exist to generate initial diversity, including geneticrecombination at multiple sites.

The BCR is composed of two genes IgH and IgK (or IgL) coding forantibody heavy and light chains. Immunoglobulins are formed byrecombination among gene segments, sequence diversification at thejunctions of these segments, and point mutations throughout the gene.Each heavy chain gene contains multiple copies of three different genesegments—a variable ‘V’ gene segment, a diversity ‘D’ gene segment, anda joining ‘J’ gene segment. Each light chain gene contains multiplecopies of two different gene segments for the variable region of theprotein—a variable ‘V’ gene segment and a joining T gene segment. Therecombination can generate a molecule with one of each of the V, D, andJ segments. Furthermore, several bases may be deleted and others added(called N and P nucleotides) at each of the two junctions, therebygenerating further diversity. After B cell activation, a process ofaffinity maturation through somatic hypermutation occurs. In thisprocess progeny cells of the activated B cells accumulate distinctsomatic mutations throughout the gene with higher mutation concentrationin the CDR regions leading to the generation of antibodies with higheraffinity to the antigens. In addition to somatic hypermutation activatedB cells undergo the process of isotype switching. Antibodies with thesame variable segments can have different forms (isotypes) depending onthe constant segment. Whereas all naïve B cells express IgM (or IgD),activated B cells mostly express IgG but also IgM, IgA and IgE. Thisexpression switching from IgM (and/or IgD) to IgG, IgA, or IgE occursthrough a recombination event causing one cell to specialize inproducing a specific isotype. A unique nucleotide sequence that arisesduring the gene arrangement process can similarly be referred to as aclonotype.

In some embodiments, the methods, compositions and systems disclosedherein are utilized to analyze the various sequences of TCRs and BCRsfrom immune cells, for example various clonotypes. In some embodiments,methods, compositions and systems disclosed herein are used to analyzethe sequence of a TCR alpha chain, a TCR beta chain, a TCR delta chain,a TCR gamma chain, or any fragment thereof (e.g., variable regionsincluding VDJ or VJ regions, constant regions, transmembrane regions,fragments thereof, combinations thereof, and combinations of fragmentsthereof). In some embodiments, methods, compositions and systemsdisclosed herein are used to analyze the sequence of a B cell receptorheavy chain, B cell receptor light chain, or any fragment thereof (e.g.,variable regions including VDJ or VJ regions, constant regions,transmembrane regions, fragments thereof, combinations thereof, andcombinations of fragments thereof).

Where immune cells are to be analyzed, primer sequences useful in any ofthe various operations for attaching barcode sequences and/oramplification reactions may comprise gene specific sequences whichtarget genes or regions of genes of immune cell proteins, for exampleimmune receptors. Such gene sequences include, but are not limited to,sequences of various T cell receptor alpha variable genes (TRAV genes),T cell receptor alpha joining genes (TRAJ genes), T cell receptor alphaconstant genes (TRAC genes), T cell receptor beta variable genes (TRBVgenes), T cell receptor beta diversity genes (TRBD genes), T cellreceptor beta joining genes (TRBJ genes), T cell receptor beta constantgenes (TRBC genes), T cell receptor gamma variable genes (TRGV genes), Tcell receptor gamma joining genes (TRGJ genes), T cell receptor gammaconstant genes (TRGC genes), T cell receptor delta variable genes (TRDVgenes), T cell receptor delta diversity genes (TRDD genes), T cellreceptor delta joining genes (TRDJ genes), and T cell receptor deltaconstant genes (TRDC genes).

MHCs, including full or partial MHC-peptides, may be used as labellingagents that are coupled to oligonucleotides that comprise a barcodesequence that identifies its associated MHC (and, thus, for example, theMHC's TCR binding partner). In some cases, MHCs are used to analyze oneor more cell-surface features of a T-cell, such as a TCR. In some cases,multiple MHCs are associated together in a larger complex to improvebinding affinity of MHCs to TCRs via multiple ligand binding synergies.

For example, as shown in FIG. 56A, the MHC peptides can individually beassociated with biotin and bound to a streptavidin moiety such that thestreptavidin moiety comprises multiple MHC moieties. Each of thesemoieties can bind to a TCR such that the streptavidin binds to thetarget T-cell via multiple MCH/TCR binding interactions. These multipleinteractions synergize and can substantially improve binding affinity.Such improved affinity can improve labelling of T-cells and also reducethe likelihood that labels will dissociate from T-cell surfaces.

As shown in FIG. 56B and continuing with this example, a barcodedoligonucleotide 5601 can be modified with streptavidin 5602 andcontacted with multiple molecules of biotinylated MHC 5604 such that theMHC 5604 molecules are coupled with the barcoded oligonucleotide 5601.The result is an MHC-complex 5608. As shown in FIG. 56B, theoligonucleotide 5601 barcode sequence 5602 can identify the MHC 5604 asits associated label and also includes sequences for hybridization withother oligonucleotides (e.g., sequence 5603 comprising a ‘Spacer C C C’and sequence 5605 comprising a ‘Spacer PCR handle’). One such otheroligonucleotide is oligonucleotide 5611 that comprises a complementarysequence 5615 (e.g., rGrGrG corresponding to C C C), a barcode sequence5613 and, such as, for example, a UMI 5614 as shown in FIG. 56C. In somecases, oligonucleotide 5611 may at first be associated with a bead andreleased from the bead. In any case, though, oligonucleotide 5611 canhybridize with oligonucleotide 5601 the MHC-oligonucleotide complex5608. The hybridized oligonucleotides 5611 and 5601 can then be extendedin primer extension reactions such that constructs comprising sequencesthat correspond to each of the two barcode sequences 5613 and 5604 aregenerated. In some cases, one or both of these corresponding sequencesmay be a complement of the original sequence in oligonucleotide 5611 or5601. One or both of the resulting constructs can be optionally furtherprocessed (e.g., to add any additional sequences and/or for clean-up)and subjected to sequencing. As described elsewhere herein, the sequencein such a construct derived from barcode sequence 5613 may be used toidentify a partition or a cell within a partition and the sequencederived from barcode sequence 5604 may be used to identify theparticular TCR on the surface of the cell, permitting a multi-assayanalysis.

Furthermore, while the example shown in FIG. 56B and FIG. 56C showsstreptavidin directly coupled to its oligonucleotide, the streptavidinmay also be coupled to a hybridization oligonucleotide which thenhybridizes with the identifying barcoded oligonucleotide, similar to theexample scheme shown in FIG. 52B (panel II) and described elsewhereherein.

The ability to characterize individual cells from larger diversepopulations of cells is also of significant value in both environmentaltesting as well as in forensic analysis, where samples may, by theirnature, be made up of diverse populations of cells and other materialthat “contaminate” the sample, relative to the cells for which thesample is being tested, e.g., environmental indicator organisms, toxicorganisms, and the like for, e.g., environmental and food safetytesting, victim and/or perpetrator cells in forensic analysis for sexualassault, and other violent crimes, and the like.

Additional useful applications of the above described single cellsequencing and characterization processes are in the field ofneuroscience research and diagnosis. In particular, neural cells caninclude long interspersed nuclear elements (LINEs), or ‘jumping’ genesthat can move around the genome, which cause each neuron to differ fromits neighbor cells. Research has shown that the number of LINEs in humanbrain exceeds that of other tissues, e.g., heart and liver tissue, withbetween 80 and 300 unique insertions (See, e.g., Coufal, N. G. et al.Nature 460, 1127-1131 (2009)). These differences have been postulated asbeing related to a person's susceptibility to neuro-logical disorders(see, e.g., Muotri, A. R. et al. Nature 468, 443-446 (2010)), or providethe brain with a diversity with which to respond to challenges. As such,the methods described herein may be used in the sequencing andcharacterization of individual neural cells.

The single cell analysis methods described herein may also be useful inthe analysis of gene expression, both in terms of identification of RNAtranscripts and their quantitation. In particular, using the single celllevel analysis methods described herein, one can isolate and analyze theRNA transcripts present in individual cells, populations of cells, orsubsets of populations of cells. In particular, in some cases, thebarcode oligonucleotides may be configured to prime, replicate andconsequently yield barcoded fragments of RNA from individual cells. Forexample, in some cases, the barcode oligonucleotides may include mRNAspecific priming sequences, e.g., poly-T primer segments that allowpriming and replication of mRNA in a reverse transcription reaction orother targeted priming sequences. Alternatively or additionally, randomRNA priming may be carried out using random N-mer primer segments of thebarcode oligonucleotides.

FIG. 6 provides a schematic of one example method for RNA expressionanalysis in individual cells using the methods described herein. Asshown, at operation 602 a cell containing sample is sorted for viablecells, which are quantified and diluted for subsequent partitioning. Atoperation 604, the individual cells separately co-partitioned with gelbeads bearing the barcoding oligonucleotides as described herein. Thecells are lysed and the barcoded oligonucleotides released into thepartitions at operation 606, where they interact with and hybridize tothe mRNA at operation 608, e.g., by virtue of a poly-T primer sequence,which is complementary to the poly-A tail of the mRNA. Using the poly-Tbarcode oligonucleotide as a priming sequence, a reverse transcriptionreaction is carried out at operation 610 to synthesize a cDNA of themRNA that includes the barcode sequence. The barcoded cDNAs are thensubjected to additional amplification at operation 612, e.g., using aPCR process, purification at operation 614, before they are placed on anucleic acid sequencing system for determination of the cDNA sequenceand its associated barcode sequence(s). In some cases, as shown,operations 602 through 608 can occur while the reagents remain in theiroriginal droplet or partition, while operations 612 through 616 canoccur in bulk (e.g., outside of the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled in order to complete operations 612through 616. In some cases, barcode oligonucleotides may be digestedwith exonucleases after the emulsion is broken. Exonuclease activity canbe inhibited by ethylenediaminetetraacetic acid (EDTA) following primerdigestion. In some cases, operation 610 may be performed either withinthe partitions based upon co-partitioning of the reverse transcriptionmixture, e.g., reverse transcriptase and associated reagents, or it maybe performed in bulk.

The structure of the barcode oligonucleotides may include a number ofsequence elements in addition to the oligonucleotide barcode sequence.One example of a barcode oligonucleotide for use in RNA analysis asdescribed above is shown in FIG. 7. As shown, the overalloligonucleotide 702 is coupled to a bead 704 by a releasable linkage706, such as a disulfide linker. The oligonucleotide may includefunctional sequences that are used in subsequent processing, such asfunctional sequence 708, which may include one or more of a sequencerspecific flow cell attachment sequence, e.g., a P5 sequence for Illuminasequencing systems, as well as sequencing primer sequences, e.g., a R1primer for Illumina sequencing systems. A barcode sequence 710 isincluded within the structure for use in barcoding the sample RNA. AnmRNA specific priming sequence, such as poly-T sequence 712 is alsoincluded in the oligonucleotide structure. An anchoring sequence segment714 may be included to ensure that the poly-T sequence hybridizes at thesequence end of the mRNA. This anchoring sequence can include a randomshort sequence of nucleotides, e.g., 1-mer, 2-mer, 3-mer or longersequence, which will ensure that the poly-T segment is more likely tohybridize at the sequence end of the poly-A tail of the mRNA. Anadditional sequence segment 716 may be provided within theoligonucleotide sequence. In some cases, this additional sequenceprovides a unique molecular identifier (UMI) sequence segment, e.g., asa random sequence (e.g., such as a random N-mer sequence) that variesacross individual oligonucleotides coupled to a single bead, whereasbarcode sequence 710 can be constant among oligonucleotides tethered toan individual bead. This unique sequence serves to provide a uniqueidentifier of the starting mRNA molecule that was captured, in order toallow quantitation of the number of original expressed RNA. As will beappreciated, although shown as a single oligonucleotide tethered to thesurface of a bead, individual bead can include tens to hundreds ofthousands or millions of individual oligonucleotide molecules (e.g., atleast about 10,000, 50,000, 100,000, 500,000, 1,000,000 or 10,000,000oligonucleotide molecules), where the barcode segment can be constant orrelatively constant for a given bead, but where the variable or uniquesequence segment will vary across an individual bead. This uniquemolecular identifier (UMI) sequence segment may include from 5 to about8 or more nucleotides within the sequence of the oligonucleotides. Insome cases, the unique molecular identifier (UMI) sequence segment canbe 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20nucleotides in length or longer. In some cases, the unique molecularidentifier (UMI) sequence segment can be at least 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length orlonger. In some cases, the unique molecular identifier (UMI) sequencesegment can be at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 nucleotides in length or shorter. In some cases,the oligonucleotide may comprise a target-specific primer. Thetarget-specific primer may bind to specific sequence in a RNA moleculeor a DNA molecule derived therefrom. For example, the specific sequencemay be a sequence that is not in the poly-A tail.

In operation, and with reference to FIGS. 6 and 7, a cell isco-partitioned along with a barcode bearing bead and lysed while thebarcoded oligonucleotides are released from the bead. The poly-T portionof the released barcode oligonucleotide then hybridizes to the poly-Atail of the mRNA. The poly-T segment then primes the reversetranscription of the mRNA to produce a cDNA of the mRNA, but whichincludes each of the sequence segments 708-716 of the barcodeoligonucleotide. Again, because the oligonucleotide 702 includes ananchoring sequence 714, it will more likely hybridize to and primereverse transcription at the sequence end of the poly-A tail of themRNA. Within any given partition, all of the cDNA of the individual mRNAmolecules will include a common barcode sequence segment 710. However,by including the unique random N-mer sequence, the transcripts made fromdifferent mRNA molecules within a given partition will vary at thisunique sequence. This provides a quantitation feature that can beidentifiable even following any subsequent amplification of the contentsof a given partition, e.g., the number of unique segments associatedwith a common barcode can be indicative of the quantity of mRNAoriginating from a single partition, and thus, a single cell. Thetranscripts may then be amplified, cleaned up and sequenced to identifythe sequence of the cDNA of the mRNA, as well as to sequence the barcodesegment and the unique sequence segment.

While a poly-T primer sequence is described, other targeted or randompriming sequences may also be used in priming the reverse transcriptionreaction. Likewise, although described as releasing the barcodedoligonucleotides into the partition along with the contents of the lysedcells, it will be appreciated that in some cases, the gel bead boundoligonucleotides may be used to hybridize and capture the mRNA on thesolid phase of the gel beads, in order to facilitate the separation ofthe RNA from other cell contents.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including messenger RNA (mRNA, including mRNA obtained from acell) analysis, is shown in FIG. 9A. As shown, the overalloligonucleotide 902 can be coupled to a bead 904 by a releasable linkage906, such as a disulfide linker. The oligonucleotide may includefunctional sequences that are used in subsequent processing, such asfunctional sequence 908, which may include a sequencer specific flowcell attachment sequence, e.g., a P5 sequence for Illumina sequencingsystems, as well as functional sequence 910, which may includesequencing primer sequences, e.g., a R1 primer binding site for Illuminasequencing systems. A barcode sequence 912 is included within thestructure for use in barcoding the sample RNA. An RNA specific (e.g.,mRNA specific) priming sequence, such as poly-T sequence 914 is alsoincluded in the oligonucleotide structure. An anchoring sequence segment(not shown) may be included to ensure that the poly-T sequencehybridizes at the sequence end of the mRNA. An additional sequencesegment 916 may be provided within the oligonucleotide sequence. Thisadditional sequence can provide a unique molecular identifier (UMI)sequence segment, e.g., as a random N-mer sequence that varies acrossindividual oligonucleotides coupled to a single bead, whereas barcodesequence 912 can be constant among oligonucleotides tethered to anindividual bead. As described elsewhere herein, this unique sequence canserve to provide a unique identifier of the starting mRNA molecule thatwas captured, in order to allow quantitation of the number of originalexpressed RNA, e.g., mRNA counting. As will be appreciated, althoughshown as a single oligonucleotide tethered to the surface of a bead,individual beads can include tens to hundreds of thousands or millionsof individual oligonucleotide molecules (e.g., at least about 10,000,50,000, 100,000, 500,000, 1,000,000 or 10,000,000 oligonucleotidemolecules), where the barcode segment can be constant or relativelyconstant for a given bead, but where the variable or unique sequencesegment will vary across an individual bead.

In an example method of cellular RNA (e.g., mRNA) analysis and inreference to FIG. 9A, a cell is co-partitioned along with a barcodebearing bead, switch oligo 924, and other reagents such as reversetranscriptase, a reducing agent and dNTPs into a partition (e.g., adroplet in an emulsion). In operation 950, the cell is lysed while thebarcoded oligonucleotides 902 are released from the bead (e.g., via theaction of the reducing agent) and the poly-T segment 914 of the releasedbarcode oligonucleotide then hybridizes to the poly-A tail of mRNA 920that is released from the cell. Next, in operation 952 the poly-Tsegment 914 is extended in a reverse transcription reaction using themRNA as a template to produce a cDNA 922 complementary to the mRNA andalso includes each of the sequence segments 908, 912, 910, 916 and 914of the barcode oligonucleotide. Terminal transferase activity of thereverse transcriptase can add additional bases to the cDNA (e.g.,polyC). The switch oligo 924 may then hybridize with the additionalbases added to the cDNA and facilitate template switching. A sequencecomplementary to the switch oligo sequence can then be incorporated intothe cDNA 922 via extension of the cDNA 922 using the switch oligo 924 asa template. Within any given partition, all of the cDNAs of theindividual mRNA molecules will include a common barcode sequence segment912. However, by including the unique random N-mer sequence 916, thetranscripts made from different mRNA molecules within a given partitionwill vary at this unique sequence. As described elsewhere herein, thisprovides a quantitation feature that can be identifiable even followingany subsequent amplification of the contents of a given partition, e.g.,the number of unique segments associated with a common barcode can beindicative of the quantity of mRNA originating from a single partition,and thus, a single cell. Following operation 952, the cDNA 922 is thenamplified with primers 926 (e.g., PCR primers) in operation 954. Next,the amplified product is then purified (e.g., via solid phase reversibleimmobilization (SPRI)) in operation 956. At operation 958, the amplifiedproduct is then sheared, ligated to additional functional sequences, andfurther amplified (e.g., via PCR). The functional sequences may includea sequencer specific flow cell attachment sequence 930, e.g., a P7sequence for Illumina sequencing systems, as well as functional sequence928, which may include a sequencing primer binding site, e.g., for a R2primer for Illumina sequencing systems, as well as functional sequence932, which may include a sample index, e.g., an i7 sample index sequencefor Illumina sequencing systems. In some cases, operations 950 and 952can occur in the partition, while operations 954, 956 and 958 can occurin bulk solution (e.g., in a pooled mixture outside of the partition).In the case where a partition is a droplet in an emulsion, the emulsioncan be broken and the contents of the droplet pooled in order tocomplete operations 954, 956 and 958. In some cases, operation 954 maybe completed in the partition. In some cases, barcode oligonucleotidesmay be digested with exonucleases after the emulsion is broken.Exonuclease activity can be inhibited by ethylenediaminetetraacetic acid(EDTA) following primer digestion. Although described in terms ofspecific sequence references used for certain sequencing systems, e.g.,Illumina systems, it will be understood that the reference to thesesequences is for illustration purposes only, and the methods describedherein may be configured for use with other sequencing systemsincorporating specific priming, attachment, index, and other operationalsequences used in those systems, e.g., systems available from IonTorrent, Oxford Nanopore, Genia, Pacific Biosciences, Complete Genomics,and the like.

In an alternative example of a barcode oligonucleotide for use in RNA(e.g., cellular RNA) analysis as shown in FIG. 9A, functional sequence908 may be a P7 sequence and functional sequence 910 may be a R2 primerbinding site. Moreover, the functional sequence 930 may be a P5sequence, functional sequence 928 may be a R1 primer binding site, andfunctional sequence 932 may be an i5 sample index sequence for Illuminasequencing systems. The configuration of the constructs generated bysuch a barcode oligonucleotide can help minimize (or avoid) sequencingof the poly-T sequence during sequencing.

Shown in FIG. 9B is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs intoa partition (e.g., a droplet in an emulsion). The switch oligo 924 maybe labeled with an additional tag 934, e.g., biotin. In operation 951,the cell is lysed while the barcoded oligonucleotides 902 (e.g., asshown in FIG. 9A) are released from the bead (e.g., via the action ofthe reducing agent). In some cases, sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site. In other cases, sequence 908is a P5 sequence and sequence 910 is a R1 primer binding site. Next, thepoly-T segment 914 of the released barcode oligonucleotide hybridizes tothe poly-A tail of mRNA 920 that is released from the cell. In operation953, the poly-T segment 914 is then extended in a reverse transcriptionreaction using the mRNA as a template to produce a cDNA 922complementary to the mRNA and also includes each of the sequencesegments 908, 912, 910, 916 and 914 of the barcode oligonucleotide.Terminal transferase activity of the reverse transcriptase can addadditional bases to the cDNA (e.g., polyC). The switch oligo 924 maythen hybridize with the cDNA and facilitate template switching. Asequence complementary to the switch oligo sequence can then beincorporated into the cDNA 922 via extension of the cDNA 922 using theswitch oligo 924 as a template. Next, an isolation operation 960 can beused to isolate the cDNA 922 from the reagents and oligonucleotides inthe partition. The additional tag 934, e.g., biotin, can be contactedwith an interacting tag 936, e.g., streptavidin, which may be attachedto a magnetic bead 938. At operation 960 the cDNA can be isolated with apull-down operation (e.g., via magnetic separation, centrifugation)before amplification (e.g., via PCR) in operation 955, followed bypurification (e.g., via solid phase reversible immobilization (SPRI)) inoperation 957 and further processing (shearing, ligation of sequences928, 932 and 930 and subsequent amplification (e.g., via PCR)) inoperation 959. In some cases where sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site, sequence 930 is a P5 sequenceand sequence 928 is a R1 primer binding site and sequence 932 is an i5sample index sequence. In some cases where sequence 908 is a P5 sequenceand sequence 910 is a R1 primer binding site, sequence 930 is a P7sequence and sequence 928 is a R2 primer binding site and sequence 932is an i7 sample index sequence. In some cases, as shown, operations 951and 953 can occur in the partition, while operations 960, 955, 957 and959 can occur in bulk solution (e.g., in a pooled mixture outside of thepartition). In the case where a partition is a droplet in an emulsion,the emulsion can be broken and the contents of the droplet pooled inorder to complete operation 960. The operations 955, 957, and 959 canthen be carried out following operation 960 after the transcripts arepooled for processing.

Shown in FIG. 9C is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs in apartition (e.g., a droplet in an emulsion). In operation 961, the cellis lysed while the barcoded oligonucleotides 902 (e.g., as shown in FIG.9A) are released from the bead (e.g., via the action of the reducingagent). In some cases, sequence 908 is a P7 sequence and sequence 910 isa R2 primer binding site. In other cases, sequence 908 is a P5 sequenceand sequence 910 is a R1 primer binding site. Next, the poly-T segment914 of the released barcode oligonucleotide then hybridizes to thepoly-A tail of mRNA 920 that is released from the cell. Next, inoperation 963 the poly-T segment 914 is then extended in a reversetranscription reaction using the mRNA as a template to produce a cDNA922 complementary to the mRNA and also includes each of the sequencesegments 908, 912, 910, 916 and 914 of the barcode oligonucleotide.Terminal transferase activity of the reverse transcriptase can addadditional bases to the cDNA (e.g., polyC). The switch oligo 924 maythen hybridize with the cDNA and facilitate template switching. Asequence complementary to the switch oligo sequence can then beincorporated into the cDNA 922 via extension of the cDNA 922 using theswitch oligo 924 as a template. Following operation 961 and operation963, mRNA 920 and cDNA 922 are denatured in operation 962. At operation964, a second strand is extended from a primer 940 having an additionaltag 942, e.g., biotin, and hybridized to the cDNA 922. Also in operation964, the biotin labeled second strand can be contacted with aninteracting tag 936, e.g., streptavidin, which may be attached to amagnetic bead 938. The cDNA can be isolated with a pull-down operation(e.g., via magnetic separation, centrifugation) before amplification(e.g., via polymerase chain reaction (PCR)) in operation 965, followedby purification (e.g., via solid phase reversible immobilization (SPRI))in operation 967 and further processing (shearing, ligation of sequences928, 932 and 930 and subsequent amplification (e.g., via PCR)) inoperation 969. In some cases where sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site, sequence 930 is a P5 sequenceand sequence 928 is a R1 primer binding site and sequence 932 is an i5sample index sequence. In some cases where sequence 908 is a P5 sequenceand sequence 910 is a R1 primer binding site, sequence 930 is a P7sequence and sequence 928 is a R2 primer binding site and sequence 932is an i7 sample index sequence. In some cases, operations 961 and 963can occur in the partition, while operations 962, 964, 965, 967, and 969can occur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled in order to complete operations 962,964, 965, 967 and 969.

Shown in FIG. 9D is another example method for RNA analysis, includingcellular mRNA analysis. In this method, the switch oligo 924 isco-partitioned with the individual cell and barcoded bead along withreagents such as reverse transcriptase, a reducing agent and dNTPs. Inoperation 971, the cell is lysed while the barcoded oligonucleotides 902(e.g., as shown in FIG. 9A) are released from the bead (e.g., via theaction of the reducing agent). In some cases, sequence 908 is a P7sequence and sequence 910 is a R2 primer binding site. In other cases,sequence 908 is a P5 sequence and sequence 910 is a R1 primer bindingsite. Next the poly-T segment 914 of the released barcodeoligonucleotide then hybridizes to the poly-A tail of mRNA 920 that isreleased from the cell. Next in operation 973, the poly-T segment 914 isthen extended in a reverse transcription reaction using the mRNA as atemplate to produce a cDNA 922 complementary to the mRNA and alsoincludes each of the sequence segments 908, 912, 910, 916 and 914 of thebarcode oligonucleotide. Terminal transferase activity of the reversetranscriptase can add additional bases to the cDNA (e.g., polyC). Theswitch oligo 924 may then hybridize with the cDNA and facilitatetemplate switching. A sequence complementary to the switch oligosequence can then be incorporated into the cDNA 922 via extension of thecDNA 922 using the switch oligo 924 as a template. In operation 966, themRNA 920, cDNA 922 and switch oligo 924 can be denatured, and the cDNA922 can be hybridized with a capture oligonucleotide 944 labeled with anadditional tag 946, e.g., biotin. In this operation, the biotin-labeledcapture oligonucleotide 944, which is hybridized to the cDNA, can becontacted with an interacting tag 936, e.g., streptavidin, which may beattached to a magnetic bead 938. Following separation from other species(e.g., excess barcoded oligonucleotides) using a pull-down operation(e.g., via magnetic separation, centrifugation), the cDNA can beamplified (e.g., via PCR) with primers 926 at operation 975, followed bypurification (e.g., via solid phase reversible immobilization (SPRI)) inoperation 977 and further processing (shearing, ligation of sequences928, 932 and 930 and subsequent amplification (e.g., via PCR)) inoperation 979. In some cases where sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site, sequence 930 is a P5 sequenceand sequence 928 is a R1 primer binding site and sequence 932 is an i5sample index sequence. In other cases where sequence 908 is a P5sequence and sequence 910 is a R1 primer binding site, sequence 930 is aP7 sequence and sequence 928 is a R2 primer binding site and sequence932 is an i7 sample index sequence. In some cases, operations 971 and973 can occur in the partition, while operations 966, 975, 977(purification), and 979 can occur in bulk (e.g., outside the partition).In the case where a partition is a droplet in an emulsion, the emulsioncan be broken and the contents of the droplet pooled in order tocomplete operations 966, 975, 977 and 979.

Shown in FIG. 9E is another example method for RNA analysis, includingcellular RNA analysis. In this method, an individual cell isco-partitioned along with a barcode bearing bead, a switch oligo 990,and other reagents such as reverse transcriptase, a reducing agent anddNTPs into a partition (e.g., a droplet in an emulsion). In operation981, the cell is lysed while the barcoded oligonucleotides (e.g., 902 asshown in FIG. 9A) are released from the bead (e.g., via the action ofthe reducing agent). In some cases, sequence 908 is a P7 sequence andsequence 910 is a R2 primer binding site. In other cases, sequence 908is a P5 sequence and sequence 910 is a R1 primer binding site. Next, thepoly-T segment of the released barcode oligonucleotide then hybridizesto the poly-A tail of mRNA 920 released from the cell. Next at operation983, the poly-T segment is then extended in a reverse transcriptionreaction to produce a cDNA 922 complementary to the mRNA and alsoincludes each of the sequence segments 908, 912, 910, 916 and 914 of thebarcode oligonucleotide. Terminal transferase activity of the reversetranscriptase can add additional bases to the cDNA (e.g., polyC). Theswitch oligo 990 may then hybridize with the cDNA and facilitatetemplate switching. A sequence complementary to the switch oligosequence and including a T7 promoter sequence, can be incorporated intothe cDNA 922. At operation 968, a second strand is synthesized and atoperation 970 the T7 promoter sequence can be used by T7 polymerase toproduce RNA transcripts in in vitro transcription. At operation 985 theRNA transcripts can be purified (e.g., via solid phase reversibleimmobilization (SPRI)), reverse transcribed to form DNA transcripts, anda second strand can be synthesized for each of the DNA transcripts. Insome cases, prior to purification, the RNA transcripts can be contactedwith a DNase (e.g., DNAase I) to break down residual DNA. At operation987 the DNA transcripts are then fragmented and ligated to additionalfunctional sequences, such as sequences 928, 932 and 930 and, in somecases, further amplified (e.g., via PCR). In some cases where sequence908 is a P7 sequence and sequence 910 is a R2 primer binding site,sequence 930 is a P5 sequence and sequence 928 is a R1 primer bindingsite and sequence 932 is an i5 sample index sequence. In some caseswhere sequence 908 is a P5 sequence and sequence 910 is a R1 primerbinding site, sequence 930 is a P7 sequence and sequence 928 is a R2primer binding site and sequence 932 is an i7 sample index sequence. Insome cases, prior to removing a portion of the DNA transcripts, the DNAtranscripts can be contacted with an RNase to break down residual RNA.In some cases, operations 981 and 983 can occur in the partition, whileoperations 968, 970, 985 and 987 can occur in bulk (e.g., outside thepartition). In the case where a partition is a droplet in an emulsion,the emulsion can be broken and the contents of the droplet pooled inorder to complete operations 968, 970, 985 and 987.

Another example of a barcode oligonucleotide for use in RNA analysis,including messenger RNA (mRNA, including mRNA obtained from a cell)analysis is shown in FIG. 10. As shown, the overall oligonucleotide 1002is coupled to a bead 1004 by a releasable linkage 1006, such as adisulfide linker. The oligonucleotide may include functional sequencesthat are used in subsequent processing, such as functional sequence1008, which may include a sequencer specific flow cell attachmentsequence, e.g., a P7 sequence, as well as functional sequence 1010,which may include sequencing primer sequences, e.g., a R2 primer bindingsite. A barcode sequence 1012 is included within the structure for usein barcoding the sample RNA. An RNA specific (e.g., mRNA specific)priming sequence, such as poly-T sequence 1014 may be included in theoligonucleotide structure. An anchoring sequence segment (not shown) maybe included to ensure that the poly-T sequence hybridizes at thesequence end of the mRNA. An additional sequence segment 1016 may beprovided within the oligonucleotide sequence. This additional sequencecan provide a unique molecular identifier (UMI) sequence segment, asdescribed elsewhere herein. An additional functional sequence 1020 maybe included for in vitro transcription, e.g., a T7 RNA polymerasepromoter sequence. As will be appreciated, although shown as a singleoligonucleotide tethered to the surface of a bead, individual beads caninclude tens to hundreds of thousands or millions of individualoligonucleotide molecules (e.g., at least about 10,000, 50,000, 100,000,500,000, 1,000,000 or 10,000,000 oligonucleotide molecules), where thebarcode segment can be constant or relatively constant for a given bead,but where the variable or unique sequence segment will vary across anindividual bead.

In an example method of cellular RNA analysis and in reference to FIG.10, a cell is co-partitioned along with a barcode bearing bead, andother reagents such as reverse transcriptase, reducing agent and dNTPsinto a partition (e.g., a droplet in an emulsion). In operation 1050,the cell is lysed while the barcoded oligonucleotides 1002 are released(e.g., via the action of the reducing agent) from the bead, and thepoly-T segment 1014 of the released barcode oligonucleotide thenhybridizes to the poly-A tail of mRNA 1020. Next at operation 1052, thepoly-T segment is then extended in a reverse transcription reactionusing the mRNA as template to produce a cDNA 1022 of the mRNA and alsoincludes each of the sequence segments 1020, 1008, 1012, 1010, 1016, and1014 of the barcode oligonucleotide. Within any given partition, all ofthe cDNAs of the individual mRNA molecules will include a common barcodesequence segment 1012. However, by including the unique random N-mersequence, the transcripts made from different mRNA molecules within agiven partition will vary at this unique sequence. As describedelsewhere herein, this provides a quantitation feature that can beidentifiable even following any subsequent amplification of the contentsof a given partition, e.g., the number of unique segments associatedwith a common barcode can be indicative of the quantity of mRNAoriginating from a single partition, and thus, a single cell. Atoperation 1054 a second strand is synthesized and at operation 1056 theT7 promoter sequence can be used by T7 polymerase to produce RNAtranscripts in in vitro transcription. At operation 1058 the transcriptsare fragmented (e.g., sheared), ligated to additional functionalsequences, and reverse transcribed. The functional sequences may includea sequencer specific flow cell attachment sequence 1030, e.g., a P5sequence, as well as functional sequence 1028, which may includesequencing primers, e.g., a R1 primer binding sequence, as well asfunctional sequence 1032, which may include a sample index, e.g., an i5sample index sequence. At operation 1060 the RNA transcripts can bereverse transcribed to DNA, the DNA amplified (e.g., via PCR), andsequenced to identify the sequence of the cDNA of the mRNA, as well asto sequence the barcode segment and the unique sequence segment. In somecases, operations 1050 and 1052 can occur in the partition, whileoperations 1054, 1056, 1058 and 1060 can occur in bulk (e.g., outsidethe partition). In the case where a partition is a droplet in anemulsion, the emulsion can be broken and the contents of the dropletpooled in order to complete operations 1054, 1056, 1058 and 1060.

In an alternative example of a barcode oligonucleotide for use in RNA(e.g., cellular RNA) analysis as shown in FIG. 10, functional sequence1008 may be a P5 sequence and functional sequence 1010 may be a R1primer binding site. Moreover, the functional sequence 1030 may be a P7sequence, functional sequence 1028 may be a R2 primer binding site, andfunctional sequence 1032 may be an i7 sample index sequence.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including messenger RNA (mRNA, including mRNA obtained from acell) analysis is shown in FIG. 11. As shown, the overalloligonucleotide 1102 is coupled to a bead 1104 by a releasable linkage1106, such as a disulfide linker. The oligonucleotide may includefunctional sequences that are used in subsequent processing, such asfunctional sequence 1108, which may include a sequencer specific flowcell attachment sequence, e.g., a P5 sequence, as well as functionalsequence 1110, which may include sequencing primer sequences, e.g., a R1primer binding site. In some cases, sequence 1108 is a P7 sequence andsequence 1110 is a R2 primer binding site. A barcode sequence 1112 isincluded within the structure for use in barcoding the sample RNA. Anadditional sequence segment 1116 may be provided within theoligonucleotide sequence. In some cases, this additional sequence canprovide a unique molecular identifier (UMI) sequence segment, asdescribed elsewhere herein. An additional sequence 1114 may be includedto facilitate template switching, e.g., polyG. As will be appreciated,although shown as a single oligonucleotide tethered to the surface of abead, individual beads can include tens to hundreds of thousands ormillions of individual oligonucleotide molecules (e.g., at least about10,000, 50,000, 100,000, 500,000, 1,000,000 or 10,000,000oligonucleotide molecules), where the barcode segment can be constant orrelatively constant for a given bead, but where the variable or uniquesequence segment will vary across an individual bead.

In an example method of cellular mRNA analysis and in reference to FIG.11, a cell is co-partitioned along with a microcapsule (e.g., beadbearing a barcoded oligonucleotide), polyT sequence, and other reagentssuch as a DNA polymerase, a reverse transcriptase, oligonucleotideprimers, dNTPs, and reducing agent into a partition (e.g., a droplet inan emulsion). The partition can serve as a reaction volume. As describedelsewhere herein, the partition serving as the reaction volume cancomprise a container or vessel such as a well, a microwell, vial, atube, through ports in nanoarray substrates, or micro-vesicles having anouter barrier surrounding an inner fluid center or core, emulsion, or adroplet. In some embodiments, the partition comprises a droplet ofaqueous fluid within a non-aqueous continuous phase, e.g., an oil phase.Within the partition, the cell can be lysed and the barcodedoligonucleotides can be released from the bead (e.g., via the action ofthe reducing agent or other stimulus). Cell lysis and release of thebarcoded oligonucleotides from the microcapsule may occur simultaneouslyin the partition (e.g., a droplet in an emulsion) or the reactionvolume. In some embodiments, cell lysis precedes release of the barcodedoligonucleotides from the microcapsule. In some embodiments, release ofthe barcoded oligonucleotides from the microcapsule precedes cell lysis.

Subsequent to cell lysis and the release of barcoded oligonucleotidesfrom the microcapsule, the reaction volume can be subjected to anamplification reaction to generate an amplification product. In anexample amplification reaction, the polyT sequence hybridizes to thepolyA tail of mRNA 1120 released from the cell as illustrated inoperation 1150. Next, in operation 1152, the polyT sequence is thenextended in a reverse transcription reaction using the mRNA as atemplate to produce a cDNA 1122 complementary to the mRNA. Terminaltransferase activity of the reverse transcriptase can add additionalbases to the cDNA (e.g., polyC) in a template independent manner. Theadditional bases added to the cDNA, e.g., polyC, can then hybridize with1114 of the barcoded oligonucleotide. This can facilitate templateswitching and a sequence complementary to the barcoded oligonucleotidecan be incorporated into the cDNA. In various embodiments, the barcodedoligonucleotide does not hybridize to the template polynucleotide.

The barcoded oligonucleotide, upon release from the microcapsule, can bepresent in the reaction volume at any suitable concentration. In someembodiments, the barcoded oligonucleotide is present in the reactionvolume at a concentration of about 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 1 μM,5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 100 μM,150 μM, 200 μM, 250 μM, 300 μM, 400 or 500 μM. In some embodiments, thebarcoded oligonucleotide is present in the reaction volume at aconcentration of at least about 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 1 μM, 5μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM, 100 μM, 150μM, 200 μM, 250 μM, 300 μM, 400 μM, 500 μM or greater. In someembodiments, the barcoded oligonucleotide is present in the reactionvolume at a concentration of at most about 0.2 μM, 0.3 μM, 0.4 μM, 0.5μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 50 μM,100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 400 μM, or 500 μM.

The transcripts can be further processed (e.g., amplified, portionsremoved, additional sequences added, etc.) and characterized asdescribed elsewhere herein. In some embodiments, the transcripts aresequenced directly. In some embodiments, the transcripts are furtherprocessed (e.g., portions removed, additional sequences added, etc) andthen sequenced. In some embodiments, the reaction volume is subjected toa second amplification reaction to generate an additional amplificationproduct. The transcripts or first amplification products can be used asthe template for the second amplification reaction. In some embodiments,primers for the second amplification reaction comprise the barcodedoligonucleotide and polyT sequence. In some embodiments, primers for thesecond amplification reaction comprise additional primers co-partitionedwith the cell. In some embodiments, these additional amplificationproducts are sequenced directly. In some embodiments, these additionalamplification products are further processed (e.g., portions removed,additional sequences added, etc) and then sequenced. The configurationof the amplification products (e.g., first amplification products andsecond amplification products) generated by such a method can helpminimize (or avoid) sequencing of the poly-T sequence during sequencing.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including cellular RNA analysis is shown in FIG. 12A. Asshown, the overall oligonucleotide 1202 is coupled to a bead 1204 by areleasable linkage 1206, such as a disulfide linker. The oligonucleotidemay include functional sequences that are used in subsequent processing,such as functional sequence 1208, which may include a sequencer specificflow cell attachment sequence, e.g., a P5 sequence, as well asfunctional sequence 1210, which may include sequencing primer sequences,e.g., a R1 primer binding site. In some cases, sequence 1208 is a P7sequence and sequence 1210 is a R2 primer binding site. A barcodesequence 1212 is included within the structure for use in barcoding thesample RNA. An additional sequence segment 1216 may be provided withinthe oligonucleotide sequence. In some cases, this additional sequencecan provide a unique molecular identifier (UMI) sequence segment, asdescribed elsewhere herein. As will be appreciated, although shown as asingle oligonucleotide tethered to the surface of a bead, individualbeads can include tens to hundreds of thousands or millions ofindividual oligonucleotide molecules (e.g., at least about 10,000,50,000, 100,000, 500,000, 1,000,000 or 10,000,000 oligonucleotidemolecules), where the barcode segment can be constant or relativelyconstant for a given bead, but where the variable or unique sequencesegment will vary across an individual bead. In an example method ofcellular RNA analysis using this barcode, a cell is co-partitioned alongwith a barcode bearing bead and other reagents such as RNA ligase and areducing agent into a partition (e.g., a droplet in an emulsion). Thecell is lysed while the barcoded oligonucleotides are released (e.g.,via the action of the reducing agent) from the bead. The barcodedoligonucleotides can then be ligated to the 5′ end of mRNA transcriptswhile in the partitions by RNA ligase. Subsequent operations may includepurification (e.g., via solid phase reversible immobilization (SPRI))and further processing (shearing, ligation of functional sequences, andsubsequent amplification (e.g., via PCR)), and these operations mayoccur in bulk (e.g., outside the partition). In the case where apartition is a droplet in an emulsion, the emulsion can be broken andthe contents of the droplet pooled for the additional operations.

An additional example of a barcode oligonucleotide for use in RNAanalysis, including cellular RNA analysis is shown in FIG. 12B. Asshown, the overall oligonucleotide 1222 is coupled to a bead 1224 by areleasable linkage 1226, such as a disulfide linker. The oligonucleotidemay include functional sequences that are used in subsequent processing,such as functional sequence 1228, which may include a sequencer specificflow cell attachment sequence, e.g., a P5 sequence, as well asfunctional sequence 1230, which may include sequencing primer sequences,e.g., a R1 primer binding site. In some cases, sequence 1228 is a P7sequence and sequence 1230 is a R2 primer binding site. A barcodesequence 1232 is included within the structure for use in barcoding thesample RNA. A priming sequence 1234 (e.g., a random priming sequence)can also be included in the oligonucleotide structure, e.g., a randomhexamer. An additional sequence segment 1236 may be provided within theoligonucleotide sequence. In some cases, this additional sequenceprovides a unique molecular identifier (UMI) sequence segment, asdescribed elsewhere herein. As will be appreciated, although shown as asingle oligonucleotide tethered to the surface of a bead, individualbeads can include tens to hundreds of thousands or millions ofindividual oligonucleotide molecules (e.g., at least about 10,000,50,000, 100,000, 500,000, 1,000,000 or 10,000,000 oligonucleotidemolecules), where the barcode segment can be constant or relativelyconstant for a given bead, but where the variable or unique sequencesegment will vary across an individual bead. In an example method ofcellular mRNA analysis using the barcode oligonucleotide of FIG. 12B, acell is co-partitioned along with a barcode bearing bead and additionalreagents such as reverse transcriptase, a reducing agent and dNTPs intoa partition (e.g., a droplet in an emulsion). The cell is lysed whilethe barcoded oligonucleotides are released from the bead (e.g., via theaction of the reducing agent). In some cases, sequence 1228 is a P7sequence and sequence 1230 is a R2 primer binding site. In other cases,sequence 1228 is a P5 sequence and sequence 1230 is a R1 primer bindingsite. The priming sequence 1234 of random hexamers can randomlyhybridize cellular mRNA. The random hexamer sequence can then beextended in a reverse transcription reaction using mRNA from the cell asa template to produce a cDNA complementary to the mRNA and also includeseach of the sequence segments 1228, 1232, 1230, 1236, and 1234 of thebarcode oligonucleotide. Subsequent operations may include purification(e.g., via solid phase reversible immobilization (SPRI)), furtherprocessing (shearing, ligation of functional sequences, and subsequentamplification (e.g., via PCR)), and these operations may occur in bulk(e.g., outside the partition). In the case where a partition is adroplet in an emulsion, the emulsion can be broken and the contents ofthe droplet pooled for additional operations. Additional reagents thatmay be co-partitioned along with the barcode bearing bead may includeoligonucleotides to block ribosomal RNA (rRNA) and nucleases to digestgenomic DNA and cDNA from cells. Alternatively, rRNA removal agents maybe applied during additional processing operations. The configuration ofthe constructs generated by such a method can help minimize (or avoid)sequencing of the poly-T sequence during sequencing.

The single cell analysis methods described herein may also be useful inthe analysis of the whole transcriptome. Referring back to the barcodeof FIG. 12B, the priming sequence 1234 may be a random N-mer. In somecases, sequence 1228 is a P7 sequence and sequence 1230 is a R2 primerbinding site. In other cases, sequence 1228 is a P5 sequence andsequence 1230 is a R1 primer binding site. In an example method of wholetranscriptome analysis using this barcode, the individual cell isco-partitioned along with a barcode bearing bead, poly-T sequence, andother reagents such as reverse transcriptase, polymerase, a reducingagent and dNTPs into a partition (e.g., droplet in an emulsion). In anoperation of this method, the cell is lysed while the barcodedoligonucleotides are released from the bead (e.g., via the action of thereducing agent) and the poly-T sequence hybridizes to the poly-A tail ofcellular mRNA. In a reverse transcription reaction using the mRNA astemplate, cDNAs of cellular mRNA can be produced. The RNA can then bedegraded with an RNase. The priming sequence 1234 in the barcodedoligonucleotide can then randomly hybridize to the cDNAs. Theoligonucleotides can be extended using polymerase enzymes and otherextension reagents co-partitioned with the bead and cell similar to asshown in FIG. 3 to generate amplification products (e.g., barcodedfragments), similar to the example amplification product shown in FIG. 3(panel F). The barcoded nucleic acid fragments may, in some casessubjected to further processing (e.g., amplification, addition ofadditional sequences, clean up processes, etc. as described elsewhereherein) characterized, e.g., through sequence analysis. In thisoperation, sequencing signals can come from full length RNA.

In an example method, the barcode sequence can be appended to the 3′ endof the template polynucleotide sequence (e.g., mRNA). Such configurationmay be useful, for example, if the sequence the 3′ end of the templatepolynucleotide is to be analyzed. In some embodiments, the barcodesequence can be appended to the 5′ end of a template polynucleotidesequence (e.g., mRNA). Such configuration may be useful, for example, ifthe sequence at the 5′ end of the template polynucleotide is to beanalyzed.

In another aspect, a partition comprises a cell co-partitioned with aprimer having a sequence towards a 3′ end that hybridizes to thetemplate polynucleotide, a template switching oligonucleotide having afirst predefined sequence towards a 5′ end, and a microcapsule, such asa bead, having barcoded oligonucleotides releasably coupled thereto. Insome embodiments, the oligonucleotides coupled to the bead includebarcode sequences that are identical (e.g., all oligonucleotides sharingthe same barcode sequence). In some aspects, the oligonucleotidescoupled to the beads additionally include unique molecular identifier(UMI) sequence segments (e.g., all oligonucleotides having differentunique molecular identifier sequences).

FIG. 18 shows a barcoded oligonucleotide coupled to a bead. As shown,the overall oligonucleotide 1802 is coupled to a bead 1804 by areleasable linkage 1806, such as a disulfide linker. The oligonucleotidemay include functional sequences that are useful for subsequentprocessing, such as functional sequence 1808, which may include asequencer specific flow cell attachment sequence, e.g., a P5 sequence,as well as functional sequence 1810, which may include sequencing primersequences, e.g., a R1 primer binding site. In some cases, sequence 1808is a P7 sequence and sequence 1810 is a R2 primer binding site. Abarcode sequence 1812 can be included within the structure for use inbarcoding the template polynucleotide. The functional sequences may beselected for compatibility with a variety of different sequencingsystems, e.g., 454 Sequencing, Ion Torrent Proton or PGM, Illumina X10,etc., and the requirements thereof. In some cases, the barcode sequence1812, functional sequences 1808 (e.g., flow cell attachment sequence)and 1810 (e.g., sequencing primer sequences) may be common to all of theoligonucleotides attached to a given bead. The barcoded oligonucleotidecan also comprise a sequence 1816 to facilitate template switching(e.g., a polyG sequence). In some cases, the additional sequenceprovides a unique molecular identifier (UMI) sequence segment, asdescribed elsewhere herein.

Although shown as a single oligonucleotide tethered to the surface of abead, individual beads can include tens to hundreds of thousands ormillions of individual oligonucleotide molecules (e.g., at least about10,000, 50,000, 100,000, 500,000, 1,000,000 or 10,000,000oligonucleotide molecules), where the barcode sequence can be constantor relatively constant for a given bead.

In an example method of cellular polynucleotide analysis using thebarcode oligonucleotide of FIG. 18, a cell is co-partitioned along witha bead bearing a barcoded oligonucleotide and additional reagents suchas reverse transcriptase, primers, oligonucleotides (e.g., templateswitching oligonucleotides), dNTPs, and reducing agent into a partition(e.g., a droplet in an emulsion). Within the partition, the cell can belysed to yield a plurality of template polynucleotides (e.g., DNA suchas genomic DNA, RNA such as mRNA, etc). In some cases, the cell is lysedusing lysis reagents that are co-partitioned with the cell.

Where the bead is a degradable or disruptable bead, the barcodedoligonucleotide can be released from the bead following the applicationof stimulus as previously described. Following release from the bead,the barcoded oligonucleotide can be present in the partition at anysuitable concentration. In some embodiments, the barcodedoligonucleotide is present in the partition at a concentration that issuitable for generating a sufficient yield of amplification products fordownstream processing and analysis, including, but not limited to,sequencing adaptor attachment and sequencing analysis. In someembodiments, the concentration of the barcoded oligonucleotide islimited by the loading capacity of the barcode bearing bead, or theamount of oligonucleotides deliverable by the bead.

The template switching oligonucleotide, which can be co-partitioned withthe cell, bead bearing barcoded oligonucleotides, etc, can be present inthe partition at any suitable concentration. In some embodiments, thetemplate switching oligonucleotide is present in the partition at aconcentration that is suitable for efficient template switching duringan amplification reaction. The concentration of the template switchingoligonucleotide can be dependent on the reagents used for dropletgeneration. In some embodiments, the template switching oligonucleotideis among a plurality of template switching oligonucleotides.

In some embodiments, the barcoded oligonucleotide and template switchingoligonucleotide are present in the partition at similar concentrations.In some embodiments, the barcoded oligonucleotide and template switchingoligonucleotides may be present in proportions reflective of the amountof amplification products to be generated using each oligonucleotide. Insome embodiments, the template switching oligonucleotide is present inthe partition at a greater concentration than the barcodedoligonucleotide. This difference in concentration can be due tolimitations on the capacity of the barcode bearing bead. In someembodiments, the concentration of the template switching oligonucleotidein the reaction volume is at least 2, 5, 10, 20, 50, 100, or 200 timesthat of the concentration of the barcoded oligonucleotide in the samereaction volume when the barcoded oligonucleotide is free in thepartition (e.g., not attached to the bead).

As illustrated in FIG. 19, a reaction mixture comprising a templatepolynucleotide from a cell 1920 and (i) the primer 1924 having asequence towards a 3′ end that hybridizes to the template polynucleotide(e.g., polyT) and (ii) a template switching oligonucleotide 1926 thatcomprises a first predefined sequence 1810 towards a 5′ end can besubjected to an amplification reaction to yield a first amplificationproduct. In some cases, the template polynucleotide is an mRNA with apolyA tail and the primer that hybridizes to the template polynucleotidecomprises a polyT sequence towards a 3′ end, which is complementary tothe polyA segment. The first predefined sequence can comprise at leastone of an adaptor sequence, a barcode sequence, a unique molecularidentifier (UMI) sequence, a primer binding site, and a sequencingprimer binding site or any combination thereof. In some cases, the firstpredefined sequence 1810 is a sequence that can be common to allpartitions of a plurality of partitions. For example, the firstpredefined sequence may comprise a flow cell attachment sequence, anamplification primer binding site, or a sequencing primer binding siteand the first amplification reaction facilitates the attachment thepredefined sequence to the template polynucleotide from the cell. Insome embodiments, the first predefined sequence comprises a primerbinding site. In some embodiments, the first predefined sequencecomprises a sequencing primer binding site. As illustrated in operation1950, the sequence towards a 3′ end (e.g., polyT) of the primer 1924hybridizes to the template polynucleotide 1920. In a first amplificationreaction, extension reaction reagents, e.g., reverse transcriptase,nucleoside triphosphates, co-factors (e.g., Mg²⁺ or Mn²⁺), that are alsoco-partitioned, can extend the primer 1924 sequence using the cell'snucleic acid as a template, to produce a transcript, e.g., cDNA, 1922having a fragment complementary to the strand of the cell's nucleic acidto which the primer annealed. In some cases, the reverse transcriptasehas terminal transferase activity and the reverse transcriptase addsadditional nucleotides, e.g., polyC, to the cDNA in a templateindependent manner. As illustrated in operation 1952, the templateswitching oligonucleotide 1926, for example a template switchingoligonucleotide which includes a polyG sequence, can hybridize to thecDNA 1922 and facilitate template switching in the first amplificationreaction. The transcript, therefore, may comprise the sequence of theprimer 1924, a sequence complementary to the template polynucleotidefrom the cell, and a sequence complementary to the template switchingoligonucleotide.

Among a plurality of partitions, each partition containing one or morecells or no cells, the primer and template switching oligonucleotide maybe universal to all partitions. Where analysis of mRNA is conducted, forexample, the primer may comprise at least a polyT segment capable ofhybridizing and priming an extension reaction from the polyA segment ofan mRNA. Where analysis of a variety of polynucleotides is conducted,the primer may comprise a random sequence capable of hybridizing to andpriming extension reactions randomly on various polynucleotidetemplates. As template switching can occur with the use of an enzymehaving terminal transferase activity, a template switchingoligonucleotide having a sequence capable of hybridizing to the appendedbases can be used for template switching in manner that is independentof the sequence of the polynucleotide templates to be analyzed. In someembodiments, the template switching oligonucleotide can comprise a firstpredefined sequence towards a 5′ end that does not specificallyhybridize to the template. In some embodiments, analysis of particulargenes is conducted. In such cases, the primer may comprise a genespecific sequence capable of hybridizing to and priming extensionreactions from templates comprising specific genes. In some embodiments,multiple genes are analyzed and a primer is among a plurality ofprimers. Each of the plurality of primers may have a sequence for aparticular gene of interest.

Subsequent to the first amplification reaction, the first amplificationproduct or transcript can be subjected to a second amplificationreaction to generate a second amplification product. In some cases,additional sequences (e.g., functional sequences such as flow cellattachment sequence, sequencing primer binding sequences, barcodesequences, etc) are to be attached. The first and second amplificationreactions can be performed in the same volume, such as for example in adroplet. In some cases, the first amplification product is subjected toa second amplification reaction in the presence of a barcodedoligonucleotide to generate a second amplification product having abarcode sequence. The barcode sequence can be unique to a partition,that is, each partition has a unique barcode sequence. The barcodedoligonucleotide may comprise a sequence of at least a segment of thetemplate switching oligonucleotide and at least a second predefinedsequence. The segment of the template switching oligonucleotide on thebarcoded oligonucleotide can facilitate hybridization of the barcodedoligonucleotide to the transcript, e.g., cDNA, to facilitate thegeneration of a second amplification product. In addition to a barcodesequence, the barcoded oligonucleotide may comprise a second definedsequence such as at least one of an adaptor sequence, a unique molecularidentifier (UMI) sequence, a primer binding site, and a sequencingprimer binding site or any combination thereof.

In some embodiments, the second amplification reaction uses the firstamplification product as a template and the barcoded oligonucleotide asa primer. As illustrated in operation 1954, the segment of the templateswitching oligonucleotide on the barcoded oligonucleotide 1928 canhybridize to the portion of the cDNA or complementary fragment 1922having a sequence complementary to the template switchingoligonucleotide or that which was copied from the template switchingoligonucleotide. In the second amplification reaction, extensionreaction reagents, e.g., polymerase, nucleoside triphosphates,co-factors (e.g., Mg²⁺ or Mn²⁺), that are also co-partitioned, canextend the primer sequence using the first amplification product astemplate as illustrated in operation 1956. The second amplificationproduct can comprise a second predefined sequence (e.g., 1808, 1812, and1810), a sequence of a segment of the template polynucleotide (e.g.,mRNA), and a sequence complementary to the primer (e.g., 1924).

In some embodiments, the second amplification product uses the barcodedoligonucleotide as a template and at least a portion of the firstamplification product as a primer. As illustrated in operation 1954, thesegment of the first amplification product (e.g., cDNA) having asequence complementary to the template switching oligonucleotide canhybridize to the segment of the barcoded oligonucleotide comprising asequence of at least a segment of the template switchingoligonucleotide. In the second amplification reaction, extensionreaction reagents, e.g., polymerase, nucleoside triphosphates,co-factors (e.g., Mg²⁺ or Mn²⁺), that are also co-partitioned, canextend the primer sequence (e.g., first amplification product) using thebarcoded oligonucleotide as template as illustrated in operation 1958.The second amplification product may comprise the sequence of the primer(e.g., 1924), a sequence which is complementary to the sequence of thetemplate polynucleotide (e.g., mRNA), and a sequence complementary tothe second predefined sequence (e.g., 1808, 1812, and 1810).

In some embodiments, the second amplification reaction is performedsubsequent to the first amplification reaction in the presence of anintervening purification operation. An intervening purificationoperation can be used, for example, to purify the template (e.g., firstamplification product) from excess reagents, including excess primerssuch as template switching oligonucleotides. In some embodiments, theamplification reaction is performed in the absence of an interveningpurification operation. In certain embodiments, an interveningpurification operation is not performed so that all sample preparationis performed in a same reaction volume. In the absence of an interveningpurification operation, the template switching oligonucleotide maycompete with barcoded oligonucleotide in the second amplificationreaction as the barcoded oligonucleotide comprises at least a segment ofthe template switching oligonucleotide. Competition between the templateswitching oligonucleotide and barcoded oligonucleotide in the secondamplification reaction to generate additional amplification product mayresult in a second amplification product lacking a barcode sequence. Insome embodiments, the template switching oligonucleotide may out-competethe barcoded oligonucleotide in the second amplification reaction if thetemplate switching oligonucleotide is present at a higher concentrationin the reaction volume than the barcoded oligonucleotide. Variousapproaches can be utilized to favor the use of the barcodedoligonucleotide in the second amplification reaction to generateamplification products having a barcode sequence in situations where thebarcoded oligonucleotide is present at a lower concentration than thetemplate switching oligonucleotide in the reaction volume.

In some embodiments, the template switching oligonucleotide is notavailable for primer extension during the second amplification reaction.In some embodiments, the template switching oligonucleotide is degradedprior to the second amplification reaction. In some embodiments, thetemplate switching oligonucleotide is degraded during the secondamplification reaction. The template switching oligonucleotide maycomprise ribonucleic acids (RNA). A template switching oligonucleotidecomprising RNA can be degraded, for example, by elevated temperatures oralkaline conditions. In some embodiments, the template switchingoligonucleotide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% RNA. In someembodiments, the template switching oligonucleotide comprises 100% RNA.In some embodiments, a first reaction rate of the second amplificationreaction using the barcoded oligonucleotide is greater than a secondreaction rate of the second amplification using the template switchingoligonucleotide.

In some embodiments, the barcoded oligonucleotide can hybridize to thefirst amplification product at a higher annealing temperature ascompared to the template switching oligonucleotide. For example, thefirst amplification product and the barcoded oligonucleotide can have ahigher melting temperature as compared to a melting temperature of thefirst amplification product and the template switching oligonucleotide.In such cases, the second amplification reaction may be performed withan annealing temperature at which the barcoded oligonucleotide is ableto hybridize to the first amplification product and initiation primerextension and at which the template switching oligonucleotide is unableto hybridize to the first amplification product and initiate primerextension. In some embodiments, the primer annealing temperature of thesecond amplification reaction is at least about 0.5° C., 1° C., 2° C.,3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C. or greater thana primer annealing temperature of the first amplification reaction. Thedifference in melting temperatures can result from the presence ofmodified nucleotides in the template switching oligonucleotide. In someembodiment, the template switching oligonucleotide comprises at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95% modified nucleotides. In some embodiments,the template switching oligonucleotide comprises 100% modifiedoligonucleotides. In some embodiments, the difference in meltingtemperature can be the result of the presence of modified nucleotides inthe barcoded oligonucleotide. In some embodiment, the barcodedoligonucleotide comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% modifiednucleotides. In some embodiments, the barcoded oligonucleotide comprises100% modified oligonucleotides. Modified nucleotides include, but arenot limited to, 2-Aminopurine, 2,6-Diaminopurine (2-Amino-dA), inverteddT, 5-Methyl dC, 2′-deoxyInosine, Super T(5-hydroxybutynl-2′-deoxyuridine), Super G (8-aza-7-deazaguanosine),locked nucleic acids (LNAs), unlocked nucleic acids (UNAs, e.g., UNA-A,UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, and 2′ Fluoro bases (e.g., FluoroC, Fluoro U, Fluoro A, and Fluoro G).

In various embodiments, the first amplification reaction is facilitatedusing an enzyme comprising polymerase activity. For example, the firstamplification reaction can be facilitated by a DNA-dependent polymeraseor a reverse-transcriptase (e.g., RNA dependent). In some embodiments,the first amplification reaction comprises polymerase chain reaction. Insome embodiments, the first amplification reaction comprises reversetranscription. In various embodiments, the second amplification reactionis facilitated using an enzyme comprising polymerase activity. Forexample, the second amplification reaction can be facilitated by aDNA-dependent polymerase. In some embodiments, the second amplificationreaction comprises polymerase chain reaction.

Following the generation of amplification products, subsequentoperations may include purification (e.g., via solid phase reversibleimmobilization (SPRI)), further processing (e.g., shearing, ligation offunctional sequences, and subsequent amplification (e.g., via PCR)).These operations may occur in bulk (e.g., outside the partition). In thecase where a partition is a droplet in an emulsion, the emulsion can bebroken and the contents of the droplet pooled for additional operations.Additional reagents that may be co-partitioned along with the barcodebearing bead may include oligonucleotides to block ribosomal RNA (rRNA)and nucleases to digest genomic DNA from cells. Alternatively, rRNAremoval agents may be applied during additional processing operations.The configuration of the constructs generated by such a method can helpminimize (or avoid) sequencing of the poly-T sequence during sequencingand/or sequence the 5′ end of a polynucleotide sequence. Theamplification products, for example first amplification products and/orsecond amplification products, may be subject to sequencing for sequenceanalysis.

Although operations with various barcode designs have been discussedindividually, individual beads can include barcode oligonucleotides ofvarious designs for simultaneous use.

In addition to characterizing individual cells or cell sub-populationsfrom larger populations, the processes and systems described herein mayalso be used to characterize individual cells as a way to provide anoverall profile of a cellular, or other organismal population. A varietyof applications require the evaluation of the presence andquantification of different cell or organism types within a populationof cells, including, for example, microbiome analysis andcharacterization, environmental testing, food safety testing,epidemiological analysis, e.g., in tracing contamination or the like. Inparticular, the analysis processes described above may be used toindividually characterize, sequence and/or identify large numbers ofindividual cells within a population. This characterization may then beused to assemble an overall profile of the originating population, whichcan provide important prognostic and diagnostic information.

For example, shifts in human microbiomes, including, e.g., gut, buccal,epidermal microbiomes, etc., have been identified as being bothdiagnostic and prognostic of different conditions or general states ofhealth. Using the single cell analysis methods and systems describedherein, one can again, characterize, sequence and identify individualcells in an overall population, and identify shifts within thatpopulation that may be indicative of diagnostic ally relevant factors.By way of example, sequencing of bacterial 16S ribosomal RNA genes hasbeen used as a highly accurate method for taxonomic classification ofbacteria. Using the targeted amplification and sequencing processesdescribed above can provide identification of individual cells within apopulation of cells. One may further quantify the numbers of differentcells within a population to identify current states or shifts in statesover time. See, e.g., Morgan et al, PLoS Comput. Biol., Ch. 12, December2012, 8(12):e1002808, and Ram et al., Syst. Biol. Reprod. Med., June2011, 57(3):162-170, each of which is entirely incorporated herein byreference for all purposes. Likewise, identification and diagnosis ofinfection or potential infection may also benefit from the single cellanalyses described herein, e.g., to identify microbial species presentin large mixes of other cells or other biological material, cells and/ornucleic acids, including the environments described above, as well asany other diagnostically relevant environments, e.g., cerebrospinalfluid, blood, fecal or intestinal samples, or the like.

The foregoing analyses may also be particularly useful in thecharacterization of potential drug resistance of different cells orpathogens, e.g., cancer cells, bacterial pathogens, etc., through theanalysis of distribution and profiling of different resistancemarkers/mutations across cell populations in a given sample.Additionally, characterization of shifts in these markers/mutationsacross populations of cells over time can provide valuable insight intothe progression, alteration, prevention, and treatment of a variety ofdiseases characterized by such drug resistance issues.

Although described in terms of cells, it will be appreciated that any ofa variety of individual biological organisms, or components of organismsare encompassed within this description, including, for example, cells,viruses, organelles, cellular inclusions, vesicles, or the like.Additionally, where referring to cells, it will be appreciated that suchreference includes any type of cell, including without limitationprokaryotic cells, eukaryotic cells, bacterial, fungal, plant,mammalian, or other animal cell types, mycoplasmas, normal tissue cells,tumor cells, or any other cell type, whether derived from single cell ormulticellular organisms.

Similarly, analysis of different environmental samples to profile themicrobial organisms, viruses, or other biological contaminants that arepresent within such samples, can provide important information aboutdisease epidemiology, and potentially aid in forecasting diseaseoutbreaks, epidemics an pandemics.

As described above, the methods, systems and compositions describedherein may also be used for analysis and characterization of otheraspects of individual cells or populations of cells.

A method 2000 for characterizing a cell is shown in FIG. 20. The method2000 may comprise, as shown in operation 2010, providing a partitioncomprising a cell and at least one labelling agent, all as describedherein. The labelling agent may be capable of binding to a cell surfacefeature of the cell, and may be coupled to a reporter oligonucleotidecomprising a nucleic acid barcode sequence that permits identificationof the labelling agent. Further, the partition may comprise one or moreanchor oligonucleotides (also referred to herein as oligonucleotides andbarcoded oligonucleotides) that are capable of interacting with thereporter oligonucleotide barcode, as described in detail herein. Next,in operation 2020, within the partition a nucleic acid moleculecomprising at least a portion of the nucleic acid barcode sequence or acomplement thereof may be synthesized, as described herein. Next, inoperation 2030, the nucleic acid molecule may be sequenced to identifythe labelling agent or the cell. In some cases, the labelling agentand/or the reporter oligonucleotide may be delivered into the cell,e.g., by transfection (e.g., using transfectamine), by lipid (e.g.,1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporterproteins.

As described herein, a labelling agent may comprise an antibody, anantibody fragment, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, a proteinscaffold, and the like, and any combination thereof. As describedherein, a cell surface feature may comprise a receptor, an antigen, asurface protein, a transmembrane protein, a cluster of differentiationprotein, a protein channel, a protein pump, a carrier protein, aphospholipid, a glycoprotein, a glycolipid, a cell-cell interactionprotein complex, an antigen-presenting complex, a majorhistocompatibility complex, an engineered T-cell receptor, a T-cellreceptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, an adherens junction., and the like, and any combinationthereof.

In some instances, prior to operation 2010, labelling agents may besubjected to conditions suitable for binding the labelling agents tocell surface features. In some instances, prior to operation 2010,labelling agents may be subjected to conditions suitable for binding thelabelling agents to cell surface features when the cell and thelabelling agents are free from the partition (e.g., prior topartitioning). In some instances, prior to operation 2010, the reporteroligonucleotide may be coupled to the labelling agent. In someinstances, in operation 2010, at least one labelling agent is bound tothe cell surface feature.

In some instances, in operation 2020, the reporter oligonucleotidecoupled to the labelling agent may be subjected to a primer extensionreaction that generates the nucleic acid molecule. In some instances, inoperation 2020, the anchor oligonucleotide may be coupled to a bead alsopartitioned with the cell and labelling agent(s), as described herein,and the method further comprises releasing the anchor oligonucleotidefrom the bead prior to synthesizing.

As described herein, the bead may comprise a gel bead. Further, asdescribed herein, the bead may comprise a diverse library of anchoroligonucleotides. In some instances, the bead may comprise at leastabout 1,000 copies of an anchor oligonucleotide, at least about 10,000copies of an anchor oligonucleotide, at least about 100,000 copies of ananchor oligonucleotide, at least about 100,000 copies of an anchoroligonucleotide, at least about 1,000,000 copies of an anchoroligonucleotide, at least about 5,000,000 copies of an anchoroligonucleotide, or at least about 10,000,000 copies of an anchoroligonucleotide. In some instances, the bead may comprise at least about1,000 copies of diverse anchor oligonucleotides, at least about 10,000copies of diverse anchor oligonucleotides, at least about 100,000 copiesof diverse anchor oligonucleotides, at least about 100,000 copies ofdiverse anchor oligonucleotides, at least about 1,00,000 copies ofdiverse anchor oligonucleotides, at least about 5,000,000 copies ofdiverse anchor oligonucleotides, or at least about 10,000,000 copies ofdiverse anchor oligonucleotides. In some instances, and as describedherein, releasing anchor oligonucleotides from the bead may comprisesubjecting the bead to a stimulus that degrades the bead. In someinstances, as described herein, releasing anchor oligonucleotides fromthe bead may comprise subjecting the bead to a chemical stimulus thatdegrades the bead.

A solid support (e.g., a bead) may comprise different types of anchoroligonucleotides for analyzing both intrinsic and extrinsic informationof a cell. For example, a solid support may comprise one or more of thefollowing: 1) an anchor oligonucleotide comprising a primer that bindsto one or more endogenous nucleic acids in the cell; 2) an anchoroligonucleotide comprising a primer that binds to one or more exogenousnucleic acids in the cell, e.g., nucleic acids from a microorganism(e.g., a virus, a bacterium) that infects the cell, nucleic acidsintroduced into the cell (e.g., such as plasmids or nucleic acid derivedtherefrom), nucleic acids for gene editing (e.g., CRISPR-related RNAsuch as crRNA, guide RNA); 3) an anchor oligonucleotide comprising aprimer that binds to a barcode (e.g., a barcode of a nucleic acid, of aprotein, or of a cell); and 4) an anchor oligonucleotide comprising asequence (e.g., a primer) that binds to a protein, e.g., an exogenousprotein expressed in the cell, an protein from a microorganism (e.g., avirus, a bacterium) that infects the cell, or an binding partner for aprotein of the cell (e.g., an antigen for an immune cell receptor).

In some cases, the methods may be used to screen cells carryingmutations, e.g., mutations generated by gene editing such as CRISPRtechnology. For example, a bead comprising a first anchoroligonucleotide with a primer for CRISPR RNA (e.g., crRNA or guide RNA)or its complementary DNA and a second anchor oligonucleotide with aprimer endogenous nucleic acid in the cell, e.g., total mRNA or aspecific mRNA. The bead may be made into a partition with a celltransfected with CRISPR RNA or a plasmid expressing CRISPR RNA. In somecases, the expressed CRISPR RNA or the plasmid may have a barcode(CRISPR barcode). The primers on the bead may be used to amplify andsequence the CRISPR RNA and endogenous mRNA, thus determining themutations generated by in the cell. In some cases, the methods may beused to perform single cell RNA sequencing, e.g., as described in Dixitet al., Perturb-Seq: Dissecting Molecular Circuits with ScalableSingle-Cell RNA Profiling of Pooled Genetic Screens Cell 167 (7),1853-1866.e17. 2016, which is incorporated herein by reference in itsentirety.

An oligonucleotide of an anchor agent or a labelling agent may comprisea backbone. The backbone may comprise one or more of the followingelements: a sequencer primer, a barcode, and a UMI. In addition to thebackbone, the oligonucleotide may also comprise a primer as describedherein, e.g., a poly-T primer, a random N-mer primer, and/or atarget-specific primer. Examples of oligonucleotides comprising variousbackbones and primer sequences are shown in FIGS. 27A-27D.

An example work flow for the methods herein may include inputting fixedreference (e.g., known transcripts from a cell with intrinsicinformation), reference templates (e.g., design of synthetic barcodes(random or target-specific) with extrinsic information, and sequencereads; and outputting classification of sequence reads as originatingfrom intrinsic or extrinsic sequences, counts of detected copies pertranscript/gene per partition, and list and counts of detected barcodesfrom extrinsic sequences per partition. In some cases, the exampleworkflow may be implemented with software.

In some instances, prior to operation 2030, the method 2000 may comprisereleasing the nucleic acid molecule from the partition (e.g., bydisruption of the partition). In some instances, operation 2030 maycomprise identifying the labelling agent (e.g., the labelling agentbound to a cell surface feature). In some instances, operation 2030 maycomprise identifying the cell surface feature from identifying thelabelling agent. In some instances, operation 2030 comprises determiningan abundance of the given cell surface feature on the cell. In someinstances, operation 2030 comprises identifying the cell. In someinstances, operation 2030 comprises identifying the labelling agent andthe cell.

In method 2000, the reporter oligonucleotide that may be coupled to thelabelling agent may comprise a unique molecular identification (UMI)sequence, as described herein. The UMI sequence may permitidentification of the cell, the labelling agent, or both. In someinstances, operation 2030 of method 2000 may comprise determining asequence of the UMI sequence and identifying the cell.

In method 2000, the anchor oligonucleotide may comprise a uniquemolecular identification (UMI) sequence, as described herein. In theseinstances, the UMI sequence of the anchor oligonucleotide may permitidentification of the cell. In some instances, operation 2030 of method2000 may comprise determining a sequence of the UMI sequence from thereporter oligonucleotide bound to the labelling agent, and a sequence ofthe UMI sequence from the anchor oligonucleotide, to identify the celland the cell surface feature.

In method 2000, and as described herein, the partition may comprise adroplet in an emulsion. In some instances, the partition comprises onlyone cell. In some instances, the cell is bound to at least one labellingagent. In some instances, the labelling agent may comprise at least twoof the same labelling agent. In some instances, the labelling agent maycomprise at least two different labelling agents. In some instances, thecell may be bound to at least about 5 different labelling agents, atleast about 10 different labelling agents, at least about 50 differentlabelling agents, at least about 100 different labelling agents, atleast about 500 different labelling agents, at least about 1,000different labelling agents, at least about 5,000 different labellingagents, at least about 10,000 different labelling agents, or at leastabout 50,000 different labelling agents. In some instances, the cell maybe bound to between about 2 and 5 different labelling agents, betweenabout 5 and 10 different labelling agents, between about 10 and 100different labelling agents, between about 100 and 500 differentlabelling agents, between about 500 and 1,000 different labellingagents, between about 1,000 and 5,000 different labelling agents,between about 5,000 and 10,000 different labelling agents, between about10,000 and 50,000 different labelling agents, or between about 2 and50,000 different labelling agents, or any range in-between. In someinstances, operation 2030 of method 2000 may comprise determining anidentity of at least a subset of the different labelling agents.

In one example process, a sample is provided that contains cells thatare to be analyzed and characterized as to their cell surface features.A cell surface feature may include, but is not limited to, a receptor,an antigen, a surface protein, a transmembrane protein, a cluster ofdifferentiation protein, a protein channel, a protein pump, a carrierprotein, a phospholipid, a glycoprotein, a glycolipid, a cell-cellinteraction protein complex, an antigen-presenting complex, a majorhistocompatibility complex, an engineered T-cell receptor, a T-cellreceptor, a B-cell receptor, a chimeric antigen receptor, a gapjunction, an adherens junction, or any combination thereof. Alsoprovided is at least one labelling agent, such as a library of labellingagents, capable of binding to a cell surface feature of interest. Alabelling agent may include, but is not limited to, an antibody, anantibody fragment, a cell surface receptor binding molecule, a receptorligand, a small molecule, a bi-specific antibody, a bi-specific T-cellengager, a T-cell receptor engager, a B-cell receptor engager, apro-body, an aptamer, a monobody, an affimer, a darpin, and a proteinscaffold, or any combination thereof. The labelling agents can include areporter oligonucleotide that is indicative of the cell surface featureto which the binding group binds. In particular, a labelling agent thatis specific to one type of cell surface feature may have coupled theretoa first reporter oligonucleotide, while a labelling agent that isspecific to a different cell surface feature may have a differentreporter oligonucleotide coupled thereto. In some aspects, thesereporter oligonucleotides may comprise nucleic acid barcode sequencesthat permit identification of the labelling agent which the reporteroligonucleotide is coupled to. The selection of oligonucleotides as thereporter may provide advantages of being able to generate significantdiversity in terms of sequence, while also being readily attachable tomost biomolecules, e.g., antibodies, etc., as well as being readilydetected, e.g., using sequencing or array technologies. In someembodiments, the labelling agents may include reporter oligonucleotidesattached to them. Thus, a first labelling agent, e.g., an antibody to afirst cell surface feature, may have associated with it a reporteroligonucleotide that has a first nucleic acid sequence. Differentlabelling agents, e.g., antibodies having binding affinity for other,different cell surface features, may have associated therewith reporteroligonucleotides that comprise different nucleic acid sequences, e.g.,having a partially or completely different nucleic acid sequence. Insome cases, for each type of cell surface feature labelling agent, e.g.,antibody or antibody fragment, the reporter oligonucleotide sequence maybe known and readily identifiable as being associated with the knowncell surface feature labelling agent. These reporter oligonucleotidesmay be directly coupled to the labelling agent, or they may be attachedto a bead, molecular lattice, e.g., a linear, globular, cross-slinked,or other polymer, or other framework that is attached or otherwiseassociated with the labelling agent, which allows attachment of multiplereporter oligonucleotides to a single labelling agent.

In the case of multiple reporter oligonucleotides coupled to a singlelabelling agent, such reporter oligonucleotides can comprise the samesequence, or a particular labelling agent may include a known set ofreporter oligonucleotide sequences. As between different labellingagents, e.g., specific for different cell surface features, the reporteroligonucleotides may be different and attributable to the particularlabelling agent.

Attachment of the reporter oligonucleotides to the labelling agents maybe achieved through any of a variety of direct or indirect, covalent ornon-covalent associations or attachments. For example, in the case ofoligonucleotide reporter oligonucleotides associated with antibody basedlabelling agents, such oligonucleotides may be covalently attached to aportion of an antibody or antibody fragment using chemical conjugationtechniques (e.g., Lightning-Link® antibody labelling kits available fromInnova Biosciences), as well as other non-covalent attachmentmechanisms, e.g., using biotinylated antibodies and oligonucleotides (orbeads that include one or more biotinylated linker, coupled tooligonucleotides) with an avidin or streptavidin linker. Antibody andoligonucleotide biotinylation techniques are available. See, e.g., Fang,et al., “Fluoride-Cleavable Biotinylation Phosphoramidite for5′-end-Labelling and Affinity Purification of SyntheticOligonucleotides,” Nucleic Acids Res. Jan. 15, 2003; 31(2):708-715,which is entirely incorporated herein by reference for all purposes.Likewise, protein and peptide biotinylation techniques have beendeveloped and are readily available. See, e.g., U.S. Pat. No. 6,265,552,which is entirely incorporated herein by reference for all purposes.Furthermore, click reaction chemistry such as a Methyltetrazine-PEG5-NHSEster reaction, a TCO-PEG4-NHS Ester reaction, or the like, may be usedto couple reporter oligonucleotides to labelling agents. In the casethat the labelling agent is a primary antibody, a reporteroligonucleotide may be coupled to the labelling agent through asecondary antibody coupling interaction. Commercially available kits,such as those from Thunderlink and Abcam, and techniques common in theart may be used to couple reporter oligonucleotides to labelling agentsas appropriate.

In some cases, a reporter oligonucleotide may be associated (e.g.,covalently linked such as conjugated or non-covalently bound through abinding interaction) to an antibody via an antibody-binding protein. Forexample, a reporter oligonucleotide and an antibody-binding protein mayform a complex. The complex may bind to a respective antibody throughthe antibody-binding protein. FIG. 28 shows an example workflow forassociating a nucleic acid (e.g., DNA) barcode on an antibody using anantibody-binding protein. An antibody binding protein 2810, e.g.,Protein A or Protein G, and an oligonucleotide comprising a nucleic acid(e.g., DNA) barcode 2820 are conjugated to the Fc region of an antibody,forming a complex 2830 comprising the antibody, the antibody-bindingprotein 2810, and the DNA barcode 2820. The complex 2830 is incubatedwith cells and unbound antibody is washed out. When the complex 2830binds to a cell, the complex and the cell are partitioned into a dropletfor further analysis.

An antibody-binding protein may have fast adsorption kinetics, slowdesorption kinetics, and/or a low binding equilibrium constant. Anymethods for adding chemical functionality to peptides or proteins may beused. Some methods may include attaching a reporter oligonucleotide tospecific amino acids or chemical groups (e.g., chemical groups presentin multiple types of proteins) on the antibody-binding protein. Theconjugation of antibody-binding proteins and oligonucleotides may beperformed using methods for forming antibody-nucleic acid conjugationdescribed herein, e.g., using click chemistry. Dissociation of theantibody-binding protein/oligonucleotide complexes may be prevented bycrosslinking (e.g., using a crosslinker such as formaldehyde), proteinengineering, or adding the protein-binding proteins in excess.

Examples of antibody-binding proteins include proteins that bind to theconstant (Fc) region of antibodies, such as Protein A, Protein G,Protein L, or fragments thereof. Other binding proteins (e.g.,streptavidin) may be expressed as fusion proteins with antibody-bindingproteins, and used to associate oligonucleotides (e.g., by binding ofbiotinylated oligonucleotides to a streptavidin-Protein A fusionprotein). Other antibody-binding proteins or domains may provideadditional binding affinity for various antibody classes. In some cases,the antibody-binding protein may be an antibody, e.g., a secondaryantibody for the antibody targeting the sample. The secondary antibodymay comprise an oligonucleotide described here, e.g., an oligonucleotidewith a barcode and a poly-A or poly T terminated sequence.

The antibody-binding proteins may be engineered to introduce additionalfunctionalities. Antibody-binding proteins may be engineered to containamino acids with functional groups amenable to conjugation witholigonucleotide. For example, the antibody-binding proteins maynaturally have or be engineered to have cysteine residues, e.g., forcontrolling stoichiometry and/or attachment location of theoligonucleotides. The antibody-binding proteins may be engineered tohave non-natural amino acid residues, e.g., for targeted crosslinking ofbinding proteins and antibodies. The antibody-binding proteins may beengineered to have tags, e.g., fluorescent tags (e.g., by fusing with afluorescent protein such as green fluorescence protein (GFP), redfluorescence protein (RFP), yellow fluorescence protein (YFP)) and/oraffinity tags for purification and visualization. The fluorescent tagsand/or the affinity tags may be cleavable. In some cases, theantigen-binding protein may be engineered to have one or more (e.g.,only one) barcode attachment sites per protein.

Also provided herein are kits comprising antibody-binding proteinsconjugated with reporter oligonucleotides, e.g., in well plates.Antibody for an assay may be incubated with the antibody-bindingproteins conjugated with reporter oligonucleotides at a specifiedconcentration without interfering with the antibody's binding siteand/or without the need for any chemistry to be carried out in thecustomer's hands to conjugate the reporter oligonucleotide to theantibody.

The reporter oligonucleotides may be provided having any of a range ofdifferent lengths, depending upon the diversity of reporteroligonucleotides suitable for a given analysis, the sequence detectionscheme employed, and the like. In some cases, these reporteroligonucleotides can be greater than or equal to about 5 nucleotides inlength, greater than or equal to about 10 nucleotides in length, greaterthan or equal to about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,150, 200 or 250 nucleotides in length. In some cases, these reporteroligonucleotides may be less than or equal to about 250, 200, 180, 150,120 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 nucleotides in length.In some cases, the reporter oligonucleotides may be selected to providebarcoded products that are already sized, and otherwise configured to beanalyzed on a sequencing system. For example, these sequences may beprovided at a length that ideally creates sequenceable products of asuitable length for particular sequencing systems. Likewise, thesereporter oligonucleotides may include additional sequence elements, inaddition to the reporter sequence, such as sequencer attachmentsequences, sequencing primer sequences, amplification primer sequences,or the complements to any of these.

In operation, a cell-containing sample may be incubated with thelabelling agents and their associated reporter oligonucleotides, for anyof the cell surface features to be analyzed. Following incubation, thecells may be washed to remove unbound labelling agents. Followingwashing, the cells may be partitioned into separate partitions, e.g.,droplets, along with the barcode (also referred to as anchoroligonucleotides) carrying beads described above, where each partitionincludes a limited number of cells, e.g., a single cell. Upon releasingof the barcodes (or anchor oligonucleotides) from the beads, they mayprime the amplification and barcoding of the reporter oligonucleotidescoupled to the labelling agents. The barcoded replicates of the reporteroligonucleotides may additionally include functional sequences, such asprimer sequences, attachment sequences or the like.

The barcoded reporter oligonucleotides may then subjected to sequenceanalysis to identify which reporter oligonucleotides were bound to thecells (i.e., cell surface features) within the partitions. Further, byalso sequencing the associated barcode sequence, one can identify that agiven cell surface feature likely came from the same cell as other,different cell surface features, whose reporter sequences include thesame barcode sequence, i.e., they were derived from the same partition.

In some embodiments, anchor oligonucleotides within the partition mayinteract with the reporter oligonucleotides coupled to labelling agentsbound to cell surface features and lead to the synthesizing of a nucleicacid molecule as described herein, where the synthesized nucleic acidmolecule may comprise at least a portion of the nucleic acid barcodesequence(s), or complement(s) thereof, that comprise the reporteroligonucleotide, or the anchor oligonucleotide, or both. Thesesynthesized nucleic acid molecules may then be subjected toamplification and sequencing, as described herein.

In some embodiments, more than one labelling agent may be bound to asingle cell surface feature, and proximity between the labelling agentsmay allow the 3′ ends of the reporter oligonucleotides coupled theretoto hybridize (wherein this hybridization is discouraged by the meltingtemperature when unbound in solution). By an extension reaction asdescribed herein, a nucleic acid molecule may be synthesized, amplified,and subjected to sequencing, as described herein.

Based upon the reporter oligonucleotides that emanate from an individualpartition based upon the presence of the barcode sequence, one may thencreate a cell surface feature profile of individual cells from apopulation of cells. Profiles of individual cells or populations ofcells may be compared to profiles from other cells, e.g., ‘normal’cells, to identify variations in cell surface features, which mayprovide diagnostically relevant information. In particular, theseprofiles may be particularly useful in the diagnosis of a variety ofdisorders that are characterized by variations in cell surfacereceptors, such as cancer and other disorders.

In some embodiments, the genomic, proteomic, and cell surfaceinformation of cells characterized by the methods and systems describedherein may be sequenced individually. In some embodiments, the genomic,proteomic, and cell surface information of cells characterized by themethods and systems described herein may be pooled and sequencedtogether. In some embodiments, the genomic, proteomic, and cell surfaceinformation of cells characterized by the methods and systems describedherein may be sequenced sequentially (i.e., cell surface informationcharacterized first, then proteomic and genomic information).

Also provided herein are compositions and methods for screening achemical compound library. The methods may comprise providing apartition comprising at least one chemical compound and an identifier ofthe partition. The identifier may be an oligonucleotide comprising anucleic acid barcode sequence as described in the application. Theidentifier oligonucleotide may be amplified and subject to sequence. Thesequence read of the identifier oligonucleotide or a fragment thereofmay be used to identify the partition and the at least one chemicalcompound in the partition. The methods may be used for screening achemical compound library in a reaction of small volumes, e.g., on thescale of nanoliters. Multiple reactions may be performed in differentpartitions with the same substrate and/or reagent. The reaction may bemultiplexed to decrease the effort and time needed to process the samenumber of compounds in reactions of larger scale, e.g., on the scale ofmicroliters. The methods and compositions may allow high throughputscreening of a chemical compound library with low noise and/orfalse-positive results. In some cases, a method for screening a chemicalcompound library may comprise one or more of the following operations:(1) providing a plurality of partitions, wherein a given partition ofthe plurality of partitions (i) has or is suspected of having at leastone chemical compound and (ii) comprises an identifier oligonucleotidecomprising a nucleic acid barcode sequence that permits identificationof the given partition; (2) subjecting the plurality of partitions toscreening under conditions sufficient to select a subset of theplurality of partitions from a remainder of the plurality of partitions,which subset comprises the given partition having or suspected of havingthe at least one chemical compound; (3) subjecting the subset of theplurality of partitions, including the given partition, to conditionssufficient to generate a nucleic acid molecule comprising at least aportion of the nucleic acid barcode sequence or a complement thereof;and (4) sequencing the nucleic acid molecule to generate sequence reads,which sequence reads permit identification of the at least one chemicalcompound.

The methods may comprise building combinatorial chemical and identifieroligonucleotide libraries on a solid support, e.g., a monodispersedpolymeric bead. The oligonucleotide barcoding may be intrinsicallylinked to a chemical synthesis path unique for that monodispersedpolymer bead. Upon partitioning this polymeric bead, the population ofcompounds may be released from the substrate to interact with the targetmolecule unencumbered by the identifier oligonucleotides. Partitions maythen be sorted based on positive/negative interactions as indicated by atraditional reporter assay. Positives partitions may then be homogenizedand pooled. The identifier oligonucleotides in the positive partitionsmay be amplified for sequencing. The methods may allow for largequantities of single compounds to be packaged into nanoliter partitionsindividually and for the subsequent deconvolution of partitions withpositive interactions that may be pooled and processed in a multiplexedformat.

In some cases, the methods comprise synthesizing a controlled number ofchemical compounds on a solid support (e.g., a bead) whilesimultaneously synthesizing a controlled number of identifieroligonucleotides unique to the compounds on the solid support. Thecombinatorial libraries of the chemical compounds and identifieroligonucleotides may be made through sequential additions of chemicalcompound subunits that concord with simultaneous or subsequentsequential additions of identifier oligonucleotides on the solid matrix.The methods may be multiplexed in a single vessel for additions ofchemical compounds and identifier oligonucleotides in a massivelyparallel way. The quantity of the chemical compounds to be screened maybe normalized.

The number of chemical compounds and/or identifier oligonucleotidessynthesized on a solid support may be controlled by adjusting the numberof attachment points. An attachment point may be a location on a solidsupport where a chemical compound or identifier oligonucleotide may beattached to. Attachment points may include multiple types of chemistriesfor the cleavage of chemical compounds and/or identifieroligonucleotides. This allows for selective release of chemicalcompounds and/or identifier oligonucleotides in a controlled fashion.The solid may have a single or multiple attachment points.

The solid support may act as a covalent linker between chemicalcompounds and identifier oligonucleotides. A single type of solidsupport or multiple types of solid support may be used in the screening.If multiple types of solid support are used, they may be covalentlylinked to form a single solid support. In certain cases, if multipletypes of solid support are used, they may be commingled (but notcovalently linked) and occupy the same physical space. A solid supportmay have two or more matrices intermingled. In these cases, chemicalcompounds and the identifier oligonucleotides may be on the same matrixor on separate matrices of the solid support. In the latter case, thechemical compounds and the identifier oligonucleotides are commingled(and not covalently linked) and occupy the same physical space. In somecases, the solid support may be permeable or non-permeable. In certaincases, the solid support may be dissolvable or non-dissolvable.

A chemical compound may be a protein (e.g., an antibody or a fragmentthereof, or an antigen or a fragment thereof), a nucleic acid molecule.In some cases, a chemical compound may be a small molecule compound. Asmall molecule compound may be a low molecular weight (e.g., no greaterthan 1000 daltons) organic compound that may help regulate a biologicalprocess. A small compound may have a size on the order of 1 nm. Forexample, a small molecule compound may be a small molecule drug.

Screening of a chemical compound library may be performed using methodsfor screening small molecules for drug discovery. For example, thescreening may be performed using high-throughput screening orhigh-content analysis in drug discovery. A high-through screening may bea screening that identifies active compounds, antibodies, or genes thatmodulate a particular biomolecular pathway. A high-content may be ascreening that identifies substances such as small molecules, peptides,or RNAi that alter the phenotype of a cell in certain manner. In somecases, a screening may be an immunoassay, e.g., enzyme-linkedimmunosorbent assay (ELISA).

Also provided herein are scaffolds for delivery of one or more reagents.In some cases, a reagent is not covalently bound to the solid scaffold.For example, the reagent may be inside the scaffold and hindered (e.g.,through steric interaction with the scaffold) from diffusing out of thescaffold. The reagent may be released from the scaffold when thescaffold is dissolved. In some cases, the scaffold may be a microcapsuledescribed herein, such as a gel bead.

The scaffold may be used in a method for characterizing a cell. Themethod may comprise providing a partition comprising a cell, a scaffold,and an reagent in the scaffold. To characterize the cell in thepartition, the scaffold may be dissolved to release the reagent. Thereagent then contacts with the cell for determining one or morecharacteristics of the cell. In some cases, the partition may comprise aplurality of reagents. Any reagent described in the disclosure may beused in this method.

The scaffold may be used to deliver two or more reagents. In some cases,a first reagent be non-covalently bound to the scaffold, and the secondreagent may be covalently bound to the scaffold. In other cases,multiple scaffolds may be used to deliver multiple reagents. In thesecases, a first reagent may be covalently bound to a first scaffold, anda second reagent may be non-covalently bound to a second scaffold. Thefirst scaffold and the second scaffold may be encapsulated in the samepartition with a cell.

The reagent that is non-covalently bound to the scaffold may be releasedwhen the scaffold is dissolved. A scaffold is dissolved when at least0.01%, 0.1, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,99%, or 100% of the volume of the scaffold is dissolved in the solutionaround it.

The scaffold may comprise one or more pores and the reagentnon-covalently bound to the scaffold may be in the one or more pores.The diameter of the one or more pores may be up to 0.01 nm, 0.1 nm, 1nm, 5 nm, 10 nm, 50 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm, 1 μm, or10 μm.

A scaffold loaded with a non-covalently bound reagent may be made usingany method of incorporating an agent in a solid substance. In somecases, the scaffold loaded with a non-covalently bound reagent may bemade using the one or more of following operations: 1) Placing thescaffold (e.g., gel bead) and the reagent under a condition that causesthe scaffold to swell and the pores defined by the polymer scaffold toenlarge. Such condition may include: in a thermodynamically-favorablesolvent, at higher or lower temperatures (e.g., fortemperature-responsive hydrogel materials), in a solvent with higher orlower ion concentration and/or in the presence or absence of an electricfield for electric charge-/field-responsive hydrogel materials; 2)Allowing sufficient time for the reagent to diffuse into the interior ofthe scaffold; 3) Transferring the scaffold into a condition that causesthe pores to shrink. The reagent molecules within the scaffold are thenhindered from diffusing out of the scaffold by steric interactions withthe polymer scaffold. The transfer in operation 3) may be achievedmicrofluidically, e.g., by moving the scaffold from one co-flowingsolvent stream to another. FIG. 29 demonstrates examples of swellingconditions and de-swelling conditions in the process. The swellabilityand pore sizes of the scaffold may be adjusted by changing the polymercomposition.

In a partition comprising a scaffold loaded with non-covalently boundreagent, the composition of the partition may be adjusted by including ascaffold of a certain volume. For example, when a partition has a fixedvolume, the concentration of the reagent in the partition may beupregulated by including a reagent-loaded scaffold of a larger volume.In some cases, the adjustment may be performed without changing theinitial concentration of the components in the partition. In certaincases, the adjustment may be performed without changing the total volumeof the partition. Such methods are useful for delivering a reagent thatinterferes with the partition generation, e.g., a cell lysis agent.

A partition with the scaffold may be generated using methods describedin the disclosure. In certain cases, during the partition generation,both the scaffold and the liquid immediately surrounding the scaffoldare encapsulated in a single partition as shown in FIG. 30. The volumeof the scaffold and the surrounding liquid comprise a “unit cell”. Unitcells may be defined by the geometry of the microchannel in whichscaffolds flow and by the pressure applied. For example, higherpressures may compress the scaffold, which are deformable, therebyreducing the volume of the unit cell.

The composition of a partition may be determined by the volume ofscaffold suspension (Z1) and the volume of the sample (Z2) encapsulatedin that partition. The characteristic of the composition may bedescribed by the ratio of these two volumes (Z1/Z2). The maximum Z1possible for single-scaffold encapsulations is equal to the volume ofthe unit cell. Thus, to increase the concentration of a reagentdelivered by the scaffold in a partition of a fixed volume withoutincreasing the concentration of the reagent in the scaffold suspension,the dimensions of the scaffold may be increased. Thus, the encapsulatedunit cell may occupy a greater volume of the partition (at higher Z1/Z2ratio). In a microchannel for making the partitions, the dimension ofthe microchannel may or may not have to be increased to accommodate thelarger partitions, depending on the mechanical properties of thescaffolds. When higher pressures are applied, the scaffold may compress,the volume of the unit cell may decrease, and a lower Z1/Z2 ratio may beachieved.

Also provided herein are methods and compositions for sequencing DNA(e.g., genomic DNA) molecules and RNA (e.g., mRNA) molecules from a cellin parallel and/or simultaneously. In some cases, the methods andcompositions may be used for sequencing the genome and transcriptomefrom a single cell in parallel. The methods may be useful to dissect thefunctional consequences of genetic variations.

A microcapsule (e.g., a bead) entrapping one or more magnetic particlesmay be used in the methods. The magnetic particles may not diffuse outof the microcapsule until the microcapsule is dissolved. Themicrocapsule may comprise an oligonucleotide comprising a DNA primer.For example, the DNA primer may be a genomic DNA primer. The DNA primermay bind to DNA molecules from a cell. The DNA primer may be used toamplify and/or sequence DNA molecules from a cell. DNA primers may beentrapped and/or bound to the microcapsule and released when themicrocapsule is dissolved.

The magnetic particles entrapped within the microcapsule may comprise anoligonucleotide comprising an RNA primer. The RNA primer may bind to RNAmolecules from a cell. In some cases, the RNA primer is an mRNA primerthat binds to the mRNA molecules from the cell. For example, the mRNAprimer may comprise a poly-T sequence that binds to the poly-A sequenceof the mRNA molecules from the cell. FIG. 31 shows a microcapsule with abarcoded magnetic particle entrapped.

The magnetic particles may be made from materials such as iron oxide(e.g., superparamagnetic iron oxide), ferromagnetic, ferrimagnetic, orparamagnetic materials. Ferromagnetic materials may be stronglysusceptible to magnetic fields and capable of retaining magneticproperties when the field can be removed. Ferromagnetic materialsinclude, but are not limited to, iron, cobalt, nickel, alloys thereof,and combinations thereof. Other ferromagnetic rare earth metals oralloys thereof can also be used to make the magnetic particles.

The oligonucleotides on both the microcapsule and the magnetic particlemay comprise the same barcode sequence. The barcode sequence may allowmatching the information (e.g., sequence reads) of DNA and RNA from thesame cell.

In some cases, the barcode sequence may comprise a unique identifier ofthe cell. For example, the unique identifier may distinguish a cell fromother cells in a sample. Thus, the unique identifier may allow parallelanalysis of DNA molecules and RNA molecules in a plurality of cells,e.g., at least 10, 50, 100, 200, 300, 400, 500, 600, 800, or 1000 cells.For example, the unique identifier may allow parallel analysis of DNAmolecules and RNA molecules in a plurality of cells, e.g., at least 200,or 500 cells.

In some cases, the microcapsule may also contain one or more reagentsfor analyzing cells. For example, the microcapsule may contain a lysisagent. When the microcapsule is dissolved, the lysis agent may bereleased and lyse the cell in the same partition with the microcapsule.

In some cases, the microcapsule may be a gel bead. An example method formaking a gel bead with one or more magnetic particles may comprise oneor more of the following operations: 1) Magnetic particles are added tothe aqueous phase of the material for making the gel beads, e.g., thegel beads monomer mixture; 2) The gel beads are made using amicrofluidic approach, e.g., by forming droplets that polymerize to formthe gel beads. When the droplets polymerize, the magnetic particles areentrapped within; 3) The same barcode sequence is added to the gel beadand the magnetic particles entrapped within, e.g., using dual ligationstrategy.

Once a partition is generated to include a cell, a microcapsule, and amagnetic particle entrapped in the microcapsule, the partition may beincubated with one or more reagents (e.g., a lysis agent) to lyse thecell and dissolve the microcapsule. The incubation may be performed on amicrofluidic chip device, e.g., with a delay line device as described inFrenz et al., Reliable microfluidic on-chip incubation of droplets indelay-lines. Lab Chip. 2009 May 21; 9(10):1344-8, which is incorporatedherein by reference in its entirety. After the incubation, the partitionmay be collected and placed in a container e.g., a strip tube or plate.

The incubation may be performed for a period that allows sufficient timefor the cell to lyse and the magnetic particles to be released from themicrocapsule. The incubation time may also allow sufficient binding ofthe RNA primers on the magnetic particles with the RNA molecules fromthe cell. In some cases, the incubation time may be from 1 minute to 100minutes, from 5 minutes to 50 minutes, from 10 minutes to 30 minutes, orfrom 10 minutes to 20 minutes.

One or more RNA molecules bound to the RNA primers on the magneticparticles may be separated from other components in the partition. Theseparation may be performed by concentrating the magnetic particles. Themagnetic particles may be concentrated by a magnetic field. Theseparation may be performed on a microfluidic device, e.g., a device asdescribed in Gao et al., Wash-free magnetic immunoassay of the PSAcancer marker using SERS and droplet microfluidics, Lab Chip, 2016, 16,1022-1029; Brouzes et al., Rapid and continuous magnetic separation indroplet microfluidic devices. Lab Chip. 2015 February 7; 15(3):908-19;or Lombardi et al., Droplet microfluidics with magnetic beads: a newtool to investigate drug-protein interactions. Anal Bioanal Chem. 2011January; 399(1):347-52, which are incorporated herein by reference intheir entireties. In some cases, the one or more RNA molecules may beseparated from DNA molecules. The separated RNA molecules and DNAmolecules from a single cell may be analyzed using approaches describedherein, e.g., sequencing, to determine a characteristic of the cell.

FIG. 32 shows a method for parallel sequencing DNA (e.g., genomic DNA)and RNA (e.g., mRNA) in a cell. In operation 3210, single cellpartitions are prepared by mixing gel beads with magnetic particles,cells and reaction reagents, e.g., a lysis agent. Droplets are generatedfrom the mixture. A single droplet 3220 contains one cell, a gel beadwith magnetic particles, and reaction reagents. The gel bead has genomicDNA primers and the magnetic particles have mRNA primers. The gel beadand the magnetic particles in the partition have the same barcodesequence. In 3230, the gel bead is dissolved to release the magneticparticles and genomic DNA primers. The cell is also lysed to release thegenomic DNA molecules and mRNA molecules. The mRNA molecules arecaptured on the magnetic particles by binding with the mRNA primers. Inoperation 3240, on a microfluidic device, the partition split into twodaughter droplets. The magnetic particles with the captured mRNAmolecules are collected in only one of the daughter droplets, thus beingseparated from other components, e.g., genomic DNA in the other daughterdroplet. Thus, the genomic DNA molecules and mRNA molecules from asingle cell are separated and may be used for further analysis.

Also provided herein are methods and compositions for analyzing one ormore proteins and one or more nucleic acids from a sample (e.g., asingle cell). For example, the methods and compositions may be used foranalyzing the proteome, and the genome or transcriptome in a singlecell. The methods may comprise generating a partition that contains thesample, a labelling agent for proteins and a labelling agent for nucleicacids. In some cases, the labelling agent for proteins may interact withone or more proteins in the sample. For example, the labelling agent forproteins may comprise an antibody. In other cases, the labelling agentfor proteins may be coupled with a protein probe that interacts with oneor more proteins in the sample. For example, the labelling agent forproteins may be coupled with an antibody. The labelling agent fornucleic acids may interact with one or more nucleic acids in the sample.The labelling agent for nucleic acids may comprise a primer, e.g., aprimer that bind to a DNA molecule and/or RNA molecule. The labellingagent for proteins and the labelling agent for nucleic acids maycomprise the same reporter oligonucleotide. The reporter oligonucleotidemay comprise a barcode and/or a UMI. The barcode and/or the UMI mayallow for matching proteins with nucleic acids from the same sample.When bound to the labelling agent for nucleic acids, the nucleic acidsfrom a sample may be sequenced. The reporter oligonucleotide or aportion thereof may also be sequenced. In some cases, the methods maycomprise one or more of the following operations: a) providing apartition comprising a biological sample comprising a protein and afirst nucleic acid molecule, a labelling agent that is (i) capable ofbinding to the protein and (ii) is coupled to a reporter oligonucleotidecomprising a nucleic acid barcode sequence that permits identificationof the labelling agent, a first anchor oligonucleotide coupled to asupport, which first anchor oligonucleotide is capable of interactingwith the reporter oligonucleotide; and a second anchor oligonucleotidecoupled to the support, which second anchor oligonucleotide is capableof interacting with the first nucleic acid molecule; (b) in thepartition, synthesizing a second nucleic acid molecule comprising atleast a portion of the nucleic acid barcode sequence or a complementthereof; and (c) subjecting the first nucleic acid molecule and thesecond nucleic acid molecule to sequencing. When the labelling agent forproteins and a protein probe is separate molecules, the protein probemay be incubated with the sample before making the partition inoperation (a).

Two anchor agents may be used in the methods. The first anchor agent mayinteract with one or more nucleic acids from a sample. Additionally oralternatively, the first anchor agent may be coupled with a labellingagent for nucleic acids. For example, the first anchor agent maycomprise an oligonucleotide that bind to a labelling agent for nucleiacid. The second anchor agent may interact with one or more proteinsfrom a sample. Additionally or alternatively, the second anchor agentmay interact be coupled with a labelling agent for proteins. Forexample, the second anchor agent may comprise an element that interactswith the labelling agent for proteins. In some cases, the second anchoragent may comprise a nucleic acid sequence that interacts with anoligonucleotide sequence coupled to a labelling agent for proteins.

The labelling agent for proteins may comprise one or more elements. Thelabelling agent may comprise an element (e.g., an oligonucleotidesequence) that interacts with an anchor agent. The labelling agent maycomprise a reporter oligonucleotide, e.g., an oligonucleotide comprisinga barcode that allows for identifying the protein targeted by thelabelling agent. For example, in the cases where the labelling agent forproteins comprises an antibody, the reporter oligonucleotide may allowfor identifying the antibody, thereby identifying the protein bound bythe antibody.

The labelling agent for proteins may comprise a reactive moiety thatallows the labelling agent to be coupled with a protein probe, e.g.,antibody. The labelling agent may be coupled with a protein probe by anychemistry descried herein for attaching a reporter oligonucleotide to alabelling agent. In some cases, the reactive moiety may include a clickchemistry linker, such as Methyltetrazine-PEG5-NHS Ester or TCO-PEG4-NHSEster. The reactive moiety on the labelling agent may also include aminefor targeting aldehydes, amine for targeting maleimide (e.g., freethiols), azide for targeting click chemistry compounds (e.g., alkynes),biotin for targeting streptavidin, phosphates for targeting EDC, whichin turn targets active ester (e.g., NH₂). The reactive moiety on theprotein probe may be a chemical compound or group that binds to thereactive moiety on the labelling agent. Example strategies to conjugatethe protein probe to the labelling agent include using of commercialkits (e.g., Solulink, Thunder link), conjugation of mild reduction ofhinge region and maleimide labelling, stain-promoted click chemistryreaction to labeled amides (e.g., copper-free), and conjugation ofperiodate oxidation of sugar chain and amine conjugation. In the caseswhere the protein probe is an antibody, the antibody may be modified forconjugating the reporter oligonucleotide. For example, the antibody maybe glycosylated with a substrate-permissive mutant ofβ-1,4-galactosyltransferase, GalT (Y289L) and azide-bearing uridinediphosphate-N-acetylgalactosamine analog uridine diphosphate-GalNAz. Themodified antibody may be conjugated with a reporter oligonucleotide witha dibenzocyclooctyne-PEG4-NHS group. FIG. 33 shows example strategiesfor antibody-reporter oligonucleotide conjugation. In some cases, somestrategy (e.g., COOH activation (e.g., EDC) and homobifunctional crosslinkers) may be avoided to prevent the protein probes from conjugatingto themselves.

The two anchor agents may be coupled to a solid support, e.g., amicrocapsule. For example, the microcapsule may be a bead, e.g., a gelbead. In some cases, the two anchor agents are coupled to the same solidsupport. In other cases, the two anchor agents are coupled to differentsolid supports. The two anchor agent may comprise the same reporteroligonucleotide.

FIG. 34 shows example reagents used in the methods. An anchor agent 3420is coupled to a bead 3410. The anchor agent comprises a barcode sequence3422 and a UMI 3423. The anchor agent also comprises an oligonucleotidesequence 3424 that allows binding to the labelling agent 3430. Thelabelling agent 3430 comprises an oligonucleotide 3431 for binding tothe anchor agent. The labelling agent 3430 also comprises a barcode 3432that allows identifying the antibody it is coupled to. The labellingagent 3430 further comprises a reactive moiety 3434 that allows thelabelling agent to couple with an antibody 3440.

An additional example of reagents and schemes suitable for analysis ofbarcoded labelling agents is shown in panels I and II of FIG. 52B. Asshown in FIG. 52B (panel I), a labelling agent (e.g., antibody, an MHCmoiety) 5201 is directly (e.g., covalently bound, bound via aprotein-protein interaction, such as with Protein G) coupled to anoligonucleotide 5202 comprising a barcode sequence 5203 that identifiesthe label agent 5201. Oligonucleotide 5202 also includes additionalsequences (sequence 5204 comprising a reverse complement of a templateswitch oligo and sequence 5205 comprising a PCR handle) suitable fordownstream reactions. FIG. 52B (panel I) also shows an additionaloligonucleotide 5206 (e.g., which may have been released from a bead asdescribed elsewhere herein) comprising a barcode sequence 5208, a UMIsequence 5209 and additional sequences (sequence 5207 comprising asequencing read primer binding site ‘pR1’ and sequence 5210 comprising atemplate switch oligo) suitable for downstream reactions. Duringanalysis, the labelling agent is bound to its target cell surfacefeature and the rGrGrG sequence of sequence 5210 hybridizes withsequence 5204 and both oligonucleotides 5202 and 5206 are extended viathe action of a polymerizing enzyme (e.g., a reverse transcriptase, apolymerase), where oligonucleotide 5206 then comprises complementsequences to oligonucleotide 5202 at its 3′ end. These constructs canthen be optionally processed as described elsewhere herein and subjectto sequencing to, for example, identify the target cell surface feature(via the complementary barcode sequence generated from oligonucleotide5202) and associate it with the cell, identified by the barcode sequenceof oligonucleotide 5206.

In another example, shown in FIG. 52B (panel II), a labelling agent(e.g., antibody) 5221 is indirectly (e.g., via hybridization) coupled toan oligonucleotide 5222 comprising a barcode sequence 5223 thatidentifies the label agent 5221. Labelling agent 5221 is directly (e.g.,covalently bound, bound via a protein-protein interaction, such as withProtein G) coupled to a hybridization oligonucleotide 5232 thathybridizes with sequence 5231 of oligonucleotide 5222. Hybridization ofoligonucleotide 5232 to oligonucleotide 5231 couples label agent 5221 tooligonucleotide 5222. Oligonucleotide 5222 also includes additionalsequences (sequence 5224 comprising a reverse complement of a templateswitch oligo and sequence 5225 comprising a PCR handle) suitable fordownstream reactions. FIG. 52B (panel II) also shows an additionaloligonucleotide 5226 (e.g., which may have been released from a bead asdescribed elsewhere herein) comprising a barcode sequence 5228, a UMIsequence 5229 and additional sequences (sequence 5227 comprising asequencing read primer binding site ‘pR1’ and sequence 5220 comprising atemplate switch oligo) suitable for downstream reactions. Duringanalysis, the labelling agent is bound to its target cell surfacefeature and the rGrGrG sequence of sequence 5220 hybridizes withsequence 5224 and both oligonucleotides 5222 and 5226 are extended viathe action of a polymerizing enzyme (e.g., a reverse transcriptase, apolymerase), where oligonucleotide 5226 then comprises complementsequences to oligonucleotide 5222 at its 3′ end. These constructs canthen be optionally processed as described elsewhere herein and subjectto sequencing to, for example, identify the target cell surface feature(via the complementary barcode sequence generated from oligonucleotide5222) and associate it with the cell, identified by the barcode sequenceof oligonucleotide 5226.

An example of the methods for analyzing mRNA molecules and proteins froma single cell is shown in FIGS. 35A and 35B. The method uses a barcodedantibody 3510 containing an antibody 3511 conjugated with anoligonucleotide 3512. The oligonucleotide 3512 can bind to a firstanchor oligonucleotide 3520 coupled to a bead. The barcoded antibody3510 is incubated with cells such that the antibody binds to an antigenon the cell, and form antibody-cell complexes (FIG. 35A). Unboundantibodies are washed out. The antibody-cell complexes are made intoemulsion partitions. Each partition contains an antibody-cell complex,the first anchor oligonucleotide 3520, and a second anchoroligonucleotide 3530 that binds to mRNA molecules from the cell. Thecell is lysed and the mRNA molecules are released from the cell. Asshown in FIG. 35B, the mRNA molecules are reverse transcribed to cDNAand amplified with the help of the second anchor oligonucleotide. Theamplified cDNA molecules have the barcode and UMI that are the same asthe barcode and UMI on the first anchor oligonucleotide 3520. Primerextension is performed on the complex of the first anchoroligonucleotide 3520 and the oligonucleotide 3512, thus generating areporter oligonucleotide 3550 comprising the barcode and UMI the same asthose on the second anchor oligonucleotide. The reporter oligonucleotide3550 also comprises an antibody identifier (antibody barcode (AbBC))that identifies the antibody and the antigen bound by the antibody. Whenthe cDNA molecules are sequenced, the sequence reads are correlated tothe antigen in the same cell using the barcode and UMI. FIG. 35C showsthe primer extension of the first anchor oligonucleotide andoligonucleotide 3512 conjugated with the antibody. The resultingoligonucleotides may be separated from cDNA synthesized from mRNA fromthe cell (e.g., by size-based selection). The first anchoroligonucleotide and the complex of the second anchor oligonucleotidewith oligonucleotide 3512 may be processed and/or sequenced separatelyor jointly. In some cases, the anchor agents 3520 and 3530 may becoupled to the same bead.

Also provided herein are the microfluidic devices used for partitioningthe cells as described above. Such microfluidic devices can comprisechannel networks for carrying out the partitioning process like thoseset forth in FIGS. 1 and 2. Briefly, these microfluidic devices cancomprise channel networks, such as those described herein, forpartitioning cells into separate partitions, and co-partitioning suchcells with oligonucleotide barcode library members, e.g., disposed onbeads. These channel networks can be disposed within a solid body, e.g.,a glass, semiconductor or polymer body structure in which the channelsare defined, where those channels communicate at their termini withreservoirs for receiving the various input fluids, and for the ultimatedeposition of the partitioned cells, etc., from the output of thechannel networks. By way of example, and with reference to FIG. 2, areservoir fluidly coupled to channel 202 may be provided with an aqueoussuspension of cells 214, while a reservoir coupled to channel 204 may beprovided with an aqueous suspension of beads 216 carrying theoligonucleotides. Channel segments 206 and 208 may be provided with anon-aqueous solution, e.g., oil, into which the aqueous fluids arepartitioned as droplets at the channel junction 212. An outlet reservoirmay be fluidly coupled to channel 210 into which the partitioned cellsand beads can be delivered and from which they may be harvested. As willbe appreciated, while described as reservoirs, it will be appreciatedthat the channel segments may be coupled to any of a variety ofdifferent fluid sources or receiving components, including tubing,manifolds, or fluidic components of other systems.

Also provided are systems that control flow of these fluids through thechannel networks e.g., through applied pressure differentials,centrifugal force, electrokinetic pumping, capillary or gravity flow, orthe like.

Also provided herein are kits for analyzing individual cells or smallpopulations of cells. The kits may include one, two, three, four, fiveor more, up to all of partitioning fluids, including both aqueousbuffers and non-aqueous partitioning fluids or oils, nucleic acidbarcode libraries that are releasably associated with beads, asdescribed herein, labelling agents, as described herein, anchoroligonucleotides, as described herein, microfluidic devices, reagentsfor disrupting cells amplifying nucleic acids, and providing additionalfunctional sequences on fragments of cellular nucleic acids orreplicates thereof, as well as instructions for using any of theforegoing in the methods described herein.

Another aspect of the disclosure provides a composition forcharacterizing a plurality of analytes, comprising a partitioncomprising a plurality of barcode molecules and the plurality ofanalytes. The plurality of barcode molecules can also include at least1,000 barcode molecules. Moreover, (i) a first individual barcodemolecule of the plurality of barcode molecules can comprise a firstnucleic acid barcode sequence that is capable of coupling to a firstanalyte of the plurality of analytes; and (ii) a second individualbarcode molecule of the plurality of barcoded molecules can comprise asecond nucleic acid barcode sequence that is capable of coupling to asecond analyte of the plurality of analytes, where the first analyte andthe second analyte are different types of analytes (e.g., DNA and RNA,DNA and protein, RNA and protein, or DNA, RNA and protein). In somecases, the composition comprises a plurality of partitions comprisingthe partition.

An additional aspect of the disclosure provides a method for analytecharacterization. The method comprises: (a) providing a plurality ofpartitions, where a given partition of the plurality of partitionscomprises a plurality of barcode molecules and a plurality of analytes.The plurality of barcode molecules can comprise at least 1,000 barcodemolecules. Moreover, (i) a first individual barcode molecule of theplurality of barcode molecules can comprise a first nucleic acid barcodesequence that is capable of coupling to a first analyte of the pluralityof analytes; and (ii) a second individual barcode molecule of theplurality of barcoded molecules can comprise a second nucleic acidbarcode sequence that is capable of coupling to a second analyte of theplurality of analytes; where the first analyte and the second analyteare different types of analytes. The method also includes (b) in saidgiven partition (i) synthesizing a first nucleic acid moleculecomprising at least a portion of the first nucleic acid barcode sequenceor complement thereof; and (ii) synthesizing a second nucleic acidmolecule comprising at least a portion of the second nucleic acidbarcode sequence or complement thereof; and (c) removing said firstnucleic acid molecule and said second molecule from said givenpartition. In some cases, the method further comprises subjecting thefirst nucleic acid molecule and the second nucleic acid molecule, or aderivative of the first nucleic acid molecule and/or second nucleic acidmolecule, to sequencing to characterize the first and/or the secondanalyte.

Characterizing the first analyte and/or the second analyte generallyprovides information regarding the first analyte and/or second analyte.This information can be used to select first and/or second analytes forone or more additional cycles of (a)-(c). Accordingly, the method mayfurther comprise repeating (a)-(c) based on a characterization of thefirst analyte or the second analyte from sequencing. In some cases, themethod further comprises selecting the first analyte and/or the secondanalyte based on a characterization of the first analyte or the secondanalyte obtained from the sequencing a subsequent sequencing uponrepeating (a)-(c).

Moreover, in some cases, (b) further comprises: (1) synthesizing thefirst nucleic acid molecule comprising at least a portion of the firstnucleic acid barcode sequence or complement thereof, and (2)synthesizing the second nucleic acid molecule comprising at least aportion of the second nucleic acid barcode sequence or complementthereof. For example, the first nucleic acid molecule and/or the secondnucleic acid molecules may be synthesized with the aid of one or moreprimer extension reactions that make use of a primer that hybridizeswith a first or second analyte. Such a primer may comprise a barcodesequence and/or a UMI sequence as described elsewhere herein. In somecases, the first nucleic acid molecule and/or the second nucleic acidmolecule may be synthesized with the aid of ligation between two nucleicacid molecules.

In some cases, the method further comprises performing one or morereactions subsequent to removing the first nucleic acid molecule and thesecond nucleic acid molecule from the given partition. Such reactionscan include the addition of additional nucleic acid sequences (e.g.,sample index sequences, a sequence for function in a particularsequencing platform) via additional primer extension reactions, nucleicacid amplification schemes (e.g., PCR) or ligation. In some cases,portions of the first and/or second nucleic acid molecules may beremoved (e.g., via restriction enzymes, via shearing) prior to or afterthe addition of additional nucleic acid sequences. Moreover, thesereactions can be performed in bulk, such that processing of the firstand second nucleic acid molecules and first and second nucleic acidmolecules from other partitions are processed simultaneously in bulk.Such processing can be completed in a single pot reaction. Examples ofsuch one or more other reactions are provided in U.S. Patent PublicationNo. 2015/0376609, which is entirely incorporated herein by reference.

An additional aspect of the disclosure provides a system forcharacterizing a plurality of analytes. The system comprises apartitioning unit for providing a partition comprising a plurality ofbarcode molecules and the plurality of analytes, where: (i) a firstindividual barcode molecule of the plurality of barcode moleculescomprises a first nucleic acid barcode sequence and is capable ofcoupling to a first analyte of the plurality of analytes; and (ii) asecond individual barcode molecule of the plurality of barcode moleculescomprises a second nucleic acid barcode sequence and is capable ofcoupling to a second analyte of the plurality of analytes, where thefirst analyte and the second analyte are different types of analytes.The system also can include a controller coupled to the partitioningunit, where the controller is programmed to: (i) direct the partitioningunit to provide the partition; subject the partition to conditions thatare sufficient to: (1) synthesize a first nucleic acid moleculecomprising at least a portion of the first nucleic acid barcode sequenceor complement thereof; and (2) synthesize a second nucleic acid moleculecomprising at least a portion of the second nucleic acid barcodesequence or complement thereof. Sequencing of the first nucleic acidmolecule and the second nucleic acid molecule, or derivatives thereof,can characterize the first analyte or the second analyte. In some cases,the partitioning unit can provides a plurality of partitions comprisingthe partition.

In some cases, the partitioning unit comprises a multi-well plate. Insome cases, the partitioning unit comprises a plurality of channels,which may be microfluidic channels. The plurality of channels may cometogether to form at least one channel junction that provides thepartition. In some cases, a partitioning unit may comprise a first (i) afirst channel fluidically connected to the at least one channel junctionand configured to provide a first fluid to the at least one channeljunction; (ii) and a second channel fluidically connected to the atleast one channel junction and configured to provide a second fluid,immiscible with the first fluid, to the at least one channel junction.In an example, then first channel may be configured to provide anaqueous phase comprising aqueous phase reagents (e.g., nucleic acids,including barcoded nucleic acids, labelling agents, beads, an agent thatcan degrade beads, amplification/primer extension reagents, samplenucleic acids, cells, cell lysis reagents, etc.) and the second channelmay be configured to provide an oil phase comprising an oil (e.g., anoil comprising a fluorosurfactant) that is immiscible with the aqueousphase. Upon contact of the aqueous phase with the oil phases, aqueousphase droplets comprising aqueous phase reagents are generated.

In various aspects, the partition or the given partition may comprise atleast 1,000 barcode molecules, at least 2,500 barcode molecules at least5,000 barcode molecules, at least 7,500 barcode molecules, at least10,000 barcode molecules, at least 20,000 barcode molecules, at least30,000 barcode molecules, at least 50,000 barcode molecules, at least60,000 barcode molecules, at least 70,000 barcode molecules, at least80,000 barcode molecules, at least 90,000 barcode molecules, at least100,000 barcode molecules, at least 200,000 barcode molecules, at least300,000 barcode molecules, at least 400,000 barcode molecules, at least500,000 barcode molecules, at least 600,000 barcode molecules, at least700,000 barcode molecules, at least 800,000 barcode molecules, at least900,000 barcode molecules, at least 1,000,000 barcode molecules, atleast 2,500,000 barcode molecules, at least 5,000,000 barcode molecules,at least 7,500,000 barcode molecules at least 10,000,000 barcodemolecules, at least 50,000,000 barcode molecules, at least 100,000,000barcode molecules or more.

In various aspects, at least one of the first individual barcodemolecule and the second individual barcode molecule may be coupled(e.g., via a covalent bond, via non-covalent interactions, via a labilebond, etc.) to a bead. In some cases, the bead comprises a gel beadand/or is degradable as described elsewhere herein. In methods describedherein, the first or second barcode molecule can be released from thebead after a partition or partitions are provided. In some cases,release of a barcode molecule may occur prior to, simultaneous to, orfollowing its use in barcoding a respective nucleic acid molecule. Whererelease happens after barcoding, barcoded constructs are initiallycoupled to the bead. Moreover, a partition may comprise an agent capableof degrading the bead. In some cases, such a reagent is a reducing agentthat can reduce disulfide bonds of the bead and/or any disulfidelinkages between species coupled to the bead and the bead itself.Moreover, in various aspects, the partition or a given partition can beany suitable partition such as a droplet among a plurality of droplets(e.g., droplets in an emulsion) or a well among a plurality of wells.Furthermore, in various aspects, the first nucleic acid barcode sequenceand the second nucleic acid barcode sequence are identical.

In various aspects, the first analyte or the second analyte can be anucleic acid molecule, including any type of nucleic acid moleculedescribed elsewhere herein. For example, the nucleic acid molecule maybe genomic deoxyribonucleic acid (gDNA). In another example, the nucleicacid molecule is messenger ribonucleic acid (mRNA).

Moreover, in various aspects, the first analyte or the second analyte isa labelling agent capable of coupling to a cell surface feature of acell. The partition or the given partition can comprise the cell or oneor more components of the cell (e.g., such as free cellular surfacefeatures remaining after cell lysis). In some cases, the partition orgiven partition comprises a single cell. The labelling agent can be anylabelling agent, including a type of labelling agent described elsewhereherein including an antibody, an antibody fragment, a cell surfacereceptor binding molecule, a receptor ligand, a small molecule, abi-specific antibody, a bi-specific T-cell engager, a T-cell receptorengager, a B-cell receptor engager, a pro-body, an aptamer, a monobody,an affimer, a darpin, a protein scaffold, an antigen, an antigenpresenting particle and a major histocompatibility complex (MHC).Examples of cell surface features include a receptor, an antigen, asurface protein, a transmembrane protein, a cluster of differentiationprotein, a protein channel, a protein pump, a carrier protein, aphospholipid, a glycoprotein, a glycolipid, a cell-cell interactionprotein complex, an antigen-presenting complex, a majorhistocompatibility complex, a T-cell receptor, a B-cell receptor, achimeric antigen receptor, a gap junction, an adherens junction and anyother cell surface feature described elsewhere herein.

In some cases, cells are incubated in bulk with one or more labellingagents prior to partitioning of cells. The one or more labelling agentscan be chosen such that they are directed to particular cell surfacefeatures of interest in a given assay. Upon binding of the one or morelabeling agents to respective cell surface features, where present, thecells can then be washed to remove unbound labelling agents and theresulting cells then subject to partitioning.

Moreover, in some cases, the first individual barcode molecule or thesecond individual barcode molecule may be capable of coupling to thelabelling agent via a third nucleic acid molecule coupled to thelabelling agent. The third nucleic acid molecule can be coupled to thelabelling agent and comprise a third nucleic acid barcode sequence thatidentifies the coupled labelling agent (and, thus, a cell surfacefeature to which the labelling agent is bound). In a primer extensionreaction, the first individual barcode molecule or the second individualbarcode molecule can be extended such that a complement of the thirdbarcode sequence is added to the first or second individual barcodemolecule. During sequencing, the first or second barcode sequence ofthese molecules can identify the partition from which the molecules weresynthesized and, where a partition comprises a single cell, the thirdbarcode sequence can associate a particular cell surface feature withthat single cell.

In various aspects, the first analyte and second analyte can bedifferent types of nucleic acid molecules. For example, the firstanalyte may be a deoxyribonucleic acid molecule (e.g., gDNA) and thesecond analyte may be ribonucleic acid molecule (e.g., mRNA), such as,for example, a transcript. Where implemented, a cell's genomic DNA andalso the cell's transcriptome can be analyzed and characterized.

Moreover, where the first and second analytes are nucleic acidmolecules, the first individual barcode molecule and/or the secondindividual barcode molecule may comprise a priming sequence capable ofhybridizing to the first analyte and/or second analyte respectively. Inaddition to the first nucleic acid barcode molecule or the secondnucleic acid barcode molecule, may also include a UMI sequence, that canbe useful for identifying (and even quantifying) particular moleculesthat are barcoded within a given partition, as is described elsewhereherein.

In an example, schematically depicted in FIG. 46A, a partition (e.g., adroplet, a well or any other type of partition described herein)comprises a bead 4601, which is coupled (e.g., reversibly coupled) tobarcoded oligonucleotides 4602 and 4603. The bead 4601 and barcodedoligonucleotides 4602 and 4603 are schematically depicted in FIG. 46A.Barcoded oligonucleotide 4602 comprises a first nucleic acid barcodesequence and a poly-T priming sequence 4604 that can hybridize with thepoly-A tail of an mRNA transcript. Barcoded oligonucleotide 4602 mayalso comprise a UMI sequence that can uniquely identify a giventranscript. Barcoded oligonucleotide 4603 comprises a second nucleicacid barcode sequence and a random N-mer priming sequence 4605 that iscapable of randomly hybridizing with gDNA. In this configuration,barcoded oligonucleotides 4602 and 4603 comprise the same nucleic acidbarcode sequence, which permits association of downstream sequencingreads with the partition. In some cases, though, the first nucleic acidbarcode sequence and the second nucleic acid barcode sequence aredifferent.

The partition also comprises a cell (not shown) and lysis agents thataid in releasing nucleic acids from the cell and can also include anagent (e.g., a reducing agent) that can degrade the bead and/or break acovalent linkage between the barcoded oligonucleotides 4602 and 4603 andbead 4601, releasing them into the partition. The released barcodedoligonucleotide 4602 can hybridize with mRNA released from the cell andthe released barcoded oligonucleotide 4603 can hybridize with gDNAreleased from the cell. Barcoded constructs A and B can then begenerated for each of the mRNA and barcoded oligonucleotide 4623 asdescribed elsewhere herein, such as via the action of a polymerase(and/or reverse transcriptase) and/or primer extension. Barcodedconstruct A can comprises a sequence corresponding to the originalbarcode sequence from the bead and a sequence corresponding to atranscript from the cell. Barcoded construct B can comprise a sequencecorresponding to the original barcode sequence from the bead and asequence corresponding to genomic DNA from the cell. The barcodedconstructs can then be released/removed from the partition and, in somecases, further processed to add any additional sequences. The resultingconstructs are then sequenced, sequencing data processed, and theresults used to characterize the mRNA and the gDNA from the cell.Analysis can be completed, for example, as described elsewhere herein.The information received from the characterization can then be used in asubsequent analysis of another cell in a partition. Moreover, barcodedoligonucleotides 4602 and 4603 can be designed to prime any particulartype of nucleic acid, including those that are not derived from a cell.Moreover, the priming sequences shown in FIG. 46A are for examplepurposes only and are not meant to be limiting.

In various aspects, the first analyte may be a nucleic acid molecule(e.g., deoxyribonucleic acid (e.g., gDNA), ribonucleic acid (e.g.,mRNA), a transcript) and the second analyte a labelling agent capable ofcoupling to a cell surface feature. In such a case, the first individualbarcode molecule may comprise a priming sequence capable of hybridizingto the nucleic acid molecule and may also include a UMI sequence.Moreover, the second individual barcode molecule may comprise a primingsequence capable of hybridizing with a third nucleic acid moleculecoupled to the labelling agent. As noted elsewhere herein, this thirdnucleic acid molecule can include a barcode sequence that identifies thelabelling agent. It may also include a UMI sequence. The labelling agentcan be any suitable labelling agent, including a type of examplelabelling agents described elsewhere herein, and may be targeted to anysuitable cell surface feature to which it can selectively bind.Non-limiting examples of such cell surface features are providedelsewhere herein. Furthermore, in some cases, the partition comprises acell having the cell surface feature and, in some cases, may compriseonly one cell.

In an example, schematically depicted in FIG. 46B, a partition (e.g., adroplet, a well, a microcapsule, or any other type of partitiondescribed herein) comprises a bead 4611, which is coupled (e.g.,reversibly coupled) to barcoded oligonucleotides 4612 and 4613. The bead4611 and barcoded oligonucleotides 4612 and 4613 are schematicallydepicted in FIG. 46B. Barcoded oligonucleotide 4612 comprises a firstnucleic acid barcode sequence and a poly-T priming sequence 4614 thatcan hybridize with the poly-A tail of an mRNA transcript. Barcodedoligonucleotide 4612 may also comprise a UMI sequence that can uniquelyidentify a given transcript. Barcoded oligonucleotide 4613 comprises asecond nucleic acid barcode sequence and a targeted priming sequencethat is capable of specifically hybridizing with a barcodedoligonucleotide 4623 (via a complementary portion 4624 of barcodedoligonucleotide 4623 coupled to an antibody 4621 that is bound to thesurface of a cell 4622. Barcoded oligonucleotide 4623 comprises abarcode sequence that uniquely identifies the antibody 4621 (and thus,the particular cell surface feature to which it is bound). In thisconfiguration, barcoded oligonucleotides 4612 and 4613 comprise the samenucleic acid barcode sequence, which permit downstream association ofbarcoded nucleic acids with the partition. In some cases, though, thefirst nucleic acid barcode sequence and the second nucleic acid barcodesequence are different. Furthermore, barcoded labelling agents,including antibodies, may be produced by any suitable route, includingvia example coupling schemes described elsewhere herein.

As shown in FIG. 46B, the partition also comprises cell 4622, lysisagents that aid in releasing nucleic acids from the cell 4622 and canalso include an agent (e.g., a reducing agent) that can degrade the beadand/or break a covalent linkage between the barcoded oligonucleotides4612 and 4613 and bead 4611, releasing them into the partition. Thereleased barcoded oligonucleotide 4612 can hybridize with mRNA releasedfrom the cell and the released barcoded oligonucleotide 4613 canhybridize with barcoded oligonucleotide 4623. Barcoded constructs A andB can then be generated for each of the mRNA and barcodedoligonucleotide 4623 as described elsewhere herein, such as via theaction of a polymerase (and/or reverse transcriptase) and/or primerextension. Barcoded construct A may comprise a sequence corresponding tothe original barcode sequence from the bead and a sequence correspondingto a transcript from the cell. Barcoded construct B may comprise asequence corresponding to the original barcode sequence from the beadand an additional sequence corresponding to the barcode sequence coupledto the labelling agent. The barcoded constructs can then bereleased/removed from the partition and, in some cases, furtherprocessed to add any additional sequences. The resulting constructs arethen sequenced, sequencing data processed, and the results used tocharacterize the mRNA and cell surface feature of the cell. Analysis,for example, can be completed as described elsewhere herein. Theinformation received from the characterization can then be used in asubsequent analysis of another cell in a partition. Moreover, thepriming sequences shown in FIG. 46B are for example purposes only andare not meant to be limiting. In addition, the scheme shown in FIG. 46Bmay also be used for concurrent analysis of genomic DNA and cell surfacefeatures. In some cases, the partition comprises only one cell.

Furthermore, in various aspects, the first analyte may comprise anucleic acid molecule with a nucleic acid sequence (mRNA, complementaryDNA derived from reverse transcription of mRNA) encoding at least aportion of a V(D)J sequence of an immune cell receptor. Accordingly, afirst barcode molecule may comprise a priming sequence that can primesuch a nucleic acid sequence, as is described elsewhere herein. In somecases, the nucleic acid molecule with a nucleic acid sequence encodingat least a portion of a V(D)J sequence of an immune cell receptor iscDNA first generated from reverse transcription of the correspondingmRNA, using a poly-T containing primer. The cDNA that is generated canthen be barcoded using a primer, comprising a barcode sequence (andoptionally, a UMI sequence) that hybridizes with at least a portion ofthe cDNA that is generated. In some cases, a template switchingoligonucleotide in conjunction a terminal transferase or a reversetranscriptase having terminal transferase activity may be employed togenerate a priming region on the cDNA to which a barcoded primer canhybridize during cDNA generation. Terminal transferase activity can, forexample, add a poly-C tail to a 3′ end of the cDNA such that thetemplate switching oligonucleotide can bind via a poly-G primingsequence and the 3′ end of the cDNA can be further extended. Theoriginal mRNA template and template switching oligonucleotide can thenbe denatured from the cDNA and the barcoded primer comprising a sequencecomplementary to at least a portion of the generated priming region onthe cDNA can then hybridize with the cDNA and a barcoded constructcomprising the barcode sequence (and any optional UMI sequence) and acomplement of the cDNA generated. Additional methods and compositionssuitable for barcoding cDNA generated from mRNA transcripts includingthose encoding V(D)J regions of an immune cell receptor and/or barcodingmethods and composition including a template switch oligonucleotide aredescribed in U.S. Provisional Patent Application Ser. No. 62/410,326,filed Oct. 19, 2016 and U.S. Provisional Patent Application Ser. No.62/490,546, filed Apr. 26, 2017, both of which applications are hereinincorporated by reference in their entireties. In one example, thescheme described elsewhere herein and schematically depicted in FIG. 19may be used for V(D)J analysis.

V(D)J analysis may also be completed with the use of one or morelabelling agents that bind to particular surface features of immunecells and are associated with barcode sequences as described elsewhereherein. In some cases, the one or more labelling agents comprise an MHC.

In some cases, different types of analytes do not include labellingagents directed to separate cell surface features of a cell.

Moreover, in various aspects, the first analyte may comprise a nucleicacid capable of functioning as a component of a gene editing reaction,such as, for example, clustered regularly interspaced short palindromicrepeats (CRISPR)-based gene editing. Accordingly, the first barcodemolecule may comprise a priming sequence that can prime such a nucleicacid sequence, as is described elsewhere herein.

While the examples described with respect to FIGS. 46A and 46B involvethe analysis of two different types of analytes, these examples are notmeant to be limiting. Any suitable number of analytes may be evaluated.Accordingly, in various aspects, there may be at least about 2, at leastabout 3, at least about 4, at least about 5, at least about 6, at leastabout 7, at least about 8, at least about 9, at least about 10, at leastabout 11, at least about 12, at least about 13, at least about 14, atleast about 15, at least about 20, at least about 25, at least about 30,at least about 40, at least about 50, at least about 100 or moredifferent analytes present in a partition, that can be subject tobarcoded sequencing analysis. Higher number, multi-assay analysis can becompleted by including primer species (one or more of which may bebarcoded) that are capable of generating barcoded constructs and capableof specifically hybridizing with a particular analyte or oligonucleotidecoupled to a labelling agent that is itself coupled to a particularanalyte in the partition and subjecting the partition to suitableconditions for barcoding.

An example reagent for multi-assay analysis is schematically depicted inFIG. 46C. As shown in FIG. 46C, a partition can include a bead 4651 thatis coupled to barcoded primers that can each participate in an assay ofa different analyte. The bead 4651 is coupled (e.g., reversibly coupled)a barcoded oligonucleotide 4652 that comprises a poly-T priming sequence4654 for mRNA analysis and is also coupled (e.g., reversibly coupled) tobarcoded oligonucleotide 4653 that comprises a random N-mer primingsequence 4655 for gDNA analysis. Moreover, bead 4651 is also coupled(e.g., reversibly coupled) to barcoded oligonucleotide 4656 that canspecifically bind an oligonucleotide coupled to a labelling agent, viaits targeted priming sequence 4657. Bead 4651 is also coupled to abarcoded oligonucleotide 4658 that can specifically bind a nucleic acidmolecule that can function in a CRISPR assay (e.g., CRISPR/Cas9), viaits targeted priming sequence 4659. In this example, each of the variousbarcoded primers comprises the same barcode sequence. Each barcodedoligonucleotide can be released from the bead 4651 within the partitionand subject to conditions suitable for analysis of its respectiveanalyte. In some cases, one or more of the analytes is associated withor derived from a cell, which itself, may be in the partition. In somecases, the partition comprises only one cell. Barcoded constructs A, B,C and D can be generated as described elsewhere herein and analyzed.Barcoded construct A may comprise a sequence corresponding to thebarcode sequence from the bead and a DNA sequence corresponding to atarget mRNA. Barcoded construct B may comprise a sequence correspondingto the barcode sequence from the bead and a sequence corresponding togenomic DNA. Barcoded construct C comprises a sequence corresponding tothe barcode sequence from the bead and a sequence corresponding tobarcode sequence associated with an antibody labelling agent. Barcodedconstruct D comprises a sequence corresponding to the barcode sequencefrom the bead and a sequence corresponding to a CRISPR nucleic acid.Each construct can be analyzed via sequencing and the results associatedwith the given cell from which the various analytes originated. Whileonly four different barcoded constructs are shown in FIG. 46C, barcoded(or even non-barcoded) constructs can be tailored for analyses of anygiven analyte associated with a nucleic acid and capable of binding withsuch a construct.

The present disclosure provides computer control systems that areprogrammed to implement methods of the disclosure, i.e., protocols ofthe disclosure. For example, the present disclosure provides computercontrol systems programmed to implement method 2000 of the presentdisclosure. FIG. 17 shows a computer system 1701 that is programmed orotherwise configured to implement methods of the disclosure includingnucleic acid sequencing methods, cell surface feature identificationmethods, interpretation of nucleic acid sequencing data and analysis ofcellular nucleic acids, such as RNA (e.g., mRNA), interpretation ofnucleic acid sequencing data and analysis of nucleic acids derived fromthe characterization of cell surface features, and characterization ofcells from sequencing data. The computer system 1701 can be anelectronic device of a user or a computer system that is remotelylocated with respect to the electronic device. The electronic device canbe a mobile electronic device.

The computer system 1701 includes a central processing unit (CPU, also“processor” and “computer processor” herein) 1705, which can be a singlecore or multi core processor, or a plurality of processors for parallelprocessing. The computer system 1701 also includes memory or memorylocation 1710 (e.g., random-access memory, read-only memory, flashmemory), electronic storage unit 1715 (e.g., hard disk), communicationinterface 1720 (e.g., network adapter) for communicating with one ormore other systems, and peripheral devices 1725, such as cache, othermemory, data storage and/or electronic display adapters. The memory1710, storage unit 1715, interface 1720 and peripheral devices 1725 arein communication with the CPU 1705 through a communication bus (solidlines), such as a motherboard. The storage unit 1715 can be a datastorage unit (or data repository) for storing data. The computer system1701 can be operatively coupled to a computer network (“network”) 1730with the aid of the communication interface 1720. The network 1730 canbe the Internet, an internet and/or extranet, or an intranet and/orextranet that is in communication with the Internet. The network 1730 insome cases is a telecommunication and/or data network. The network 1730can include one or more computer servers, which can enable distributedcomputing, such as cloud computing. The network 1730, in some cases withthe aid of the computer system 1701, can implement a peer-to-peernetwork, which may enable devices coupled to the computer system 1701 tobehave as a client or a server.

The CPU 1705 can execute a sequence of machine-readable instructions,which can be embodied in a program or software. The instructions may bestored in a memory location, such as the memory 1710. The instructionscan be directed to the CPU 1705, which can subsequently program orotherwise configure the CPU 1705 to implement methods of the presentdisclosure. Examples of operations performed by the CPU 1705 can includefetch, decode, execute, and writeback.

The CPU 1705 can be part of a circuit, such as an integrated circuit.One or more other components of the system 1701 can be included in thecircuit. In some cases, the circuit is an application specificintegrated circuit (ASIC).

The storage unit 1715 can store files, such as drivers, libraries andsaved programs. The storage unit 1715 can store user data, e.g., userpreferences and user programs. The computer system 1701 in some casescan include one or more additional data storage units that are externalto the computer system 1701, such as located on a remote server that isin communication with the computer system 1701 through an intranet orthe Internet.

The computer system 1701 can communicate with one or more remotecomputer systems through the network 1730. For instance, the computersystem 1701 can communicate with a remote computer system of a user.Examples of remote computer systems include personal computers (e.g.,portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® GalaxyTab), telephones, Smart phones (e.g., Apple® iPhone, Android-enableddevice, Blackberry®), or personal digital assistants. The user canaccess the computer system 1701 via the network 1730.

Methods as described herein can be implemented by way of machine (e.g.,computer processor) executable code stored on an electronic storagelocation of the computer system 1701, such as, for example, on thememory 1710 or electronic storage unit 1715. The machine executable ormachine readable code can be provided in the form of software. Duringuse, the code can be executed by the processor 1705. In some cases, thecode can be retrieved from the storage unit 1715 and stored on thememory 1710 for ready access by the processor 1705. In some situations,the electronic storage unit 1715 can be precluded, andmachine-executable instructions are stored on memory 1710.

The code can be pre-compiled and configured for use with a machinehaving a processer adapted to execute the code, or can be compiledduring runtime. The code can be supplied in a programming language thatcan be selected to enable the code to execute in a pre-compiled oras-compiled fashion.

Aspects of the systems and methods provided herein, such as the computersystem 1701, can be embodied in programming. Various aspects of thetechnology may be thought of as “products” or “articles of manufacture”typically in the form of machine (or processor) executable code and/orassociated data that is carried on or embodied in a type of machinereadable medium. Machine-executable code can be stored on an electronicstorage unit, such as memory (e.g., read-only memory, random-accessmemory, flash memory) or a hard disk. “Storage” type media can includeany or all of the tangible memory of the computers, processors or thelike, or associated modules thereof, such as various semiconductormemories, tape drives, disk drives and the like, which may providenon-transitory storage at any time for the software programming. All orportions of the software may at times be communicated through theInternet or various other telecommunication networks. Suchcommunications, for example, may enable loading of the software from onecomputer or processor into another, for example, from a managementserver or host computer into the computer platform of an applicationserver. Thus, another type of media that may bear the software elementsincludes optical, electrical and electromagnetic waves, such as usedacross physical interfaces between local devices, through wired andoptical landline networks and over various air-links. The physicalelements that carry such waves, such as wired or wireless links, opticallinks or the like, also may be considered as media bearing the software.As used herein, unless restricted to non-transitory, tangible “storage”media, terms such as computer or machine “readable medium” refer to anymedium that participates in providing instructions to a processor forexecution.

Hence, a machine readable medium, such as computer-executable code, maytake many forms, including but not limited to, a tangible storagemedium, a carrier wave medium or physical transmission medium.Non-volatile storage media include, for example, optical or magneticdisks, such as any of the storage devices in any computer(s) or thelike, such as may be used to implement the databases, etc. shown in thedrawings. Volatile storage media include dynamic memory, such as mainmemory of such a computer platform. Tangible transmission media includecoaxial cables; copper wire and fiber optics, including the wires thatcomprise a bus within a computer system. Carrier-wave transmission mediamay take the form of electric or electromagnetic signals, or acoustic orlight waves such as those generated during radio frequency (RF) andinfrared (IR) data communications. Common forms of computer-readablemedia therefore include for example: a floppy disk, a flexible disk,hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD orDVD-ROM, any other optical medium, punch cards paper tape, any otherphysical storage medium with patterns of holes, a RAM, a ROM, a PROM andEPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wavetransporting data or instructions, cables or links transporting such acarrier wave, or any other medium from which a computer may readprogramming code and/or data. Many of these forms of computer readablemedia may be involved in carrying one or more sequences of one or moreinstructions to a processor for execution.

The computer system 1701 can include or be in communication with anelectronic display screen 1735 that comprises a user interface (UI) 1740for providing, for example, results of nucleic acid sequencing, analysisof nucleic acid sequencing data, characterization of nucleic acidsequencing samples, cell characterizations, etc. Examples of UI'sinclude, without limitation, a graphical user interface (GUI) andweb-based user interface. The system 1701 may comprise an electronicdisplay screen 1735 comprising a user interface 1740 that displays agraphical element that is accessible by a user to execute a protocol perthe methods described herein, (e.g. to characterize cells), and acomputer processor coupled to the electronic display screen andprogrammed to execute the protocol upon selection of the graphicalelement by the user.

Methods and systems of the present disclosure can be implemented by wayof one or more algorithms. An algorithm can be implemented by way ofsoftware upon execution by the central processing unit 1705. Thealgorithm can, for example, initiate nucleic acid sequencing, processnucleic acid sequencing data, interpret nucleic acid sequencing results,characterize nucleic acid samples, characterize cells, etc.

Barcoded oligonucleotides as described elsewhere herein may be generatedin any suitable manner and comprise one or more sequences in addition toa barcode sequence. As noted elsewhere herein, one such sequence can bea priming sequence that can aid in barcoding analytes. Moreover, abarcoded oligonucleotide may also comprise one or more additionalfunctional sequences that may, for example, aid in rendering thebarcoded oligonucleotide compatible with a given sequencing platform(e.g., functional sequences may be flow cell adaptor immobilizationsequences (such as, for example, P7 and P5 from an Illumina platform),sequencing primer binding site sequences (such as, for example, R1 froman Illumina platform), and other priming sites for downstreamamplification, such as, for example, a Nextera functional sequence or aTruSeq functional sequence.

In some cases, barcoded oligonucleotides are coupled to beads and beadsmay comprise oligonucleotides having a first type functional sequence ata given position and oligonucleotides having a second, different type offunctional sequence at the given position. An example is depicted inFIG. 50A. As shown in FIG. 50A, a bead may be coupled tooligonucleotides comprising a TruSeq functional sequence and also tooligonucleotides comprising a Nextera functional sequence. Onto each ofthese sequences additional sequences can be added to generate a fulloligonucleotide also comprising a nucleic acid barcode sequence, anoptional UMI sequence and a priming sequence. Attachment of thesesequences can be via ligation (including via splint ligation as isdescribed in U.S. Patent Publication No. 20140378345, which is hereinincorporated by reference in its entirety) or any other suitable route.Sequences of example barcoded oligonucleotides comprising a TruSeqfunctional group are shown in FIG. 50B and sequences of example barcodedoligonucleotides comprising a Nextera functional group are shown in FIG.50C. Each of the example barcoded oligonucleotides shown in FIG. 50B andFIG. 50B (top sequence for each construct) are shown hybridized withsplint sequences (bottom sequence for each construct) that can behelpful in constructing complete barcoded oligonucleotides.

In some aspects, methods provided herein may also be used to preparepolynucleotide contained within cells in a manner that enablescell-specific information to be obtained. The methods enable detectionof genetic variations (e.g., SNPs, mutations, indels, copy numbervariations, transversions, translocations, inversions, etc.) from verysmall samples, such as from samples comprising about 10-100 cells. Insome cases, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cellsmay be used in the methods described herein. In some cases, at leastabout 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be usedin the methods described herein. In other cases, at most about 5, 10,20, 30, 40, 50, 60, 70, 80, 90 or 100 cells may be used in the methodsdescribed herein.

In an example, a method comprises partitioning a cellular sample (orcrude cell extract) such that at most one cell (or extract of one cell)is present per partition, lysing the cells, fragmenting thepolynucleotides contained within the cells by any of the methodsdescribed herein, attaching the fragmented polynucleotides to barcodes,pooling, and sequencing.

The barcodes and other reagents may be contained within a microcapsule.These microcapsules may be loaded into a partition (e.g., a microwell, adroplet) before, after, or concurrently with the loading of the cell,such that each cell is contacted with a different microcapsule. Thistechnique may be used to attach a unique barcode to polynucleotidesobtained from each cell. The resulting tagged polynucleotides may thenbe pooled and sequenced, and the barcodes may be used to trace theorigin of the polynucleotides. For example, polynucleotides withidentical barcodes may be determined to originate from the same cell,while polynucleotides with different barcodes may be determined tooriginate from different cells.

The methods described herein may be used to detect the distribution ofoncogenic mutations across a population of cancerous tumor cells. Forexample, some tumor cells may have a mutation, or amplification, of anoncogene (e.g., HER2, BRAF, EGFR, KRAS) in both alleles (homozygous),others may have a mutation in one allele (heterozygous), and stillothers may have no mutation (wild-type). The methods described hereinmay be used to detect these differences, and also to quantify therelative numbers of homozygous, heterozygous, and wild-type cells. Suchinformation may be used, for example, to stage a particular cancerand/or to monitor the progression of the cancer and its treatment overtime.

In some examples, this disclosure provides methods of identifyingmutations in two different oncogenes (e.g., KRAS and EGFR). If the samecell comprises genes with both mutations, this may indicate a moreaggressive form of cancer. In contrast, if the mutations are located intwo different cells, this may indicate that the cancer is more benign,or less advanced.

EXAMPLES Example I: Cellular RNA Analysis Using Emulsions

In an example, reverse transcription with template switching and cDNAamplification (via PCR) is performed in emulsion droplets withoperations as shown in FIG. 9A. The reaction mixture that is partitionedfor reverse transcription and cDNA amplification (via PCR) includes1,000 cells or 10,000 cells or 10 ng of RNA, beads bearing barcodedoligonucleotides/0.2% Tx-100/5× Kapa buffer, 2× Kapa HS HiFi Ready Mix,4 μM switch oligo, and Smartscribe. Where cells are present, the mixtureis partitioned such that a majority or all of the droplets comprise asingle cell and single bead. The cells are lysed while the barcodedoligonucleotides are released from the bead, and the poly-T segment ofthe barcoded oligonucleotide hybridizes to the poly-A tail of mRNA thatis released from the cell as in operation 950. The poly-T segment isextended in a reverse transcription reaction as in operation 952 and thecDNA is amplified as in operation 954. The thermal cycling conditionsare 42° C. for 130 minutes; 98° C. for 2 min; and 35 cycles of thefollowing 98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 6 min.Following thermal cycling, the emulsion is broken and the transcriptsare purified with Dynabeads and 0.6×SPRI as in operation 956.

The yield from template switch reverse transcription and PCR inemulsions is shown for 1,000 cells in FIG. 13A and 10,000 cells in FIG.13C and 10 ng of RNA in FIG. 13B (Smartscribe line). The cDNAs from RTand PCR performed in emulsions for 10 ng RNA is sheared and ligated tofunctional sequences, cleaned up with 0.8×SPRI, and is further amplifiedby PCR as in operation 958. The amplification product is cleaned up with0.8×SPRI. The yield from this processing is shown in FIG. 13B (SSIIline).

Example II: Cellular RNA Analysis Using Emulsions

In another example, reverse transcription with template switching andcDNA amplification (via PCR) is performed in emulsion droplets withoperations as shown in FIG. 9A. The reaction mixture that is partitionedfor reverse transcription and cDNA amplification (via PCR) includesJurkat cells, beads bearing barcoded oligonucleotides/0.2%TritonX-100/5× Kapa buffer, 2× Kapa HS HiFi Ready Mix, 4 μM switcholigo, and Smartscribe. The mixture is partitioned such that a majorityor all of the droplets comprise a single cell and single bead. The cellsare lysed while the barcoded oligonucleotides are released from thebead, and the poly-T segment of the barcoded oligonucleotide hybridizesto the poly-A tail of mRNA that is released from the cell as inoperation 950. The poly-T segment is extended in a reverse transcriptionreaction as in operation 952 and the cDNA is amplified as in operation954. The thermal cycling conditions are 42° C. for 130 minutes; 98° C.for 2 min; and 35 cycles of the following 98° C. for 15 sec, 60° C. for20 sec, and 72° C. for 6 min. Following thermal cycling, the emulsion isbroken and the transcripts are cleaned-up with Dynabeads and 0.6×SPRI asin operation 956. The yield from reactions with various cell numbers(625 cells, 1,250 cells, 2,500 cells, 5,000 cells, and 10,000 cells) isshown in FIG. 14A. These yields are confirmed with GADPH qPCR assayresults shown in FIG. 14B.

Example III: RNA Analysis Using Emulsions

In another example, reverse transcription is performed in emulsiondroplets and cDNA amplification is performed in bulk in a manner similarto that as shown in FIG. 9C. The reaction mixture that is partitionedfor reverse transcription includes beads bearing barcodedoligonucleotides, 10 ng Jurkat RNA (e.g., Jurkat mRNA), 5× First-Strandbuffer, and Smartscribe. The barcoded oligonucleotides are released fromthe bead, and the poly-T segment of the barcoded oligonucleotidehybridizes to the poly-A tail of the RNA as in operation 961. The poly-Tsegment is extended in a reverse transcription reaction as in operation963. The thermal cycling conditions for reverse transcription are onecycle at 42° C. for 2 hours and one cycle at 70° C. for 10 min.Following thermal cycling, the emulsion is broken and RNA and cDNAs aredenatured as in operation 962. A second strand is then synthesized byprimer extension with a primer having a biotin tag as in operation 964.The reaction conditions for this primer extension include cDNA as thefirst strand and biotinylated extension primer ranging in concentrationfrom 0.5-3.0 μM. The thermal cycling conditions are one cycle at 98° C.for 3 min and one cycle of 98° C. for 15 sec, 60° C. for 20 sec, and 72°C. for 30 min. Following primer extension, the second strand is pulleddown with Dynabeads MyOne Streptavidin C1 and T1, and cleaned-up withAgilent SureSelect XT buffers. The second strand is pre-amplified viaPCR as in operation 965 with the following cycling conditions—one cycleat 98° C. for 3 min and one cycle of 98° C. for 15 sec, 60° C. for 20sec, and 72° C. for 30 min. The yield for various concentrations ofbiotinylated primer (0.5 μM, 1.0 μM, 2.0 μM, and 3.0 μM) is shown inFIG. 15.

Example IV: RNA Analysis Using Emulsions

In another example, in vitro transcription by T7 polymerase is used toproduce RNA transcripts as shown in FIG. 10. The mixture that ispartitioned for reverse transcription includes beads bearing barcodedoligonucleotides which also include a T7 RNA polymerase promotersequence, 10 ng human RNA (e.g., human mRNA), 5 x First-Strand buffer,and Smartscribe. The mixture is partitioned such that a majority or allof the droplets comprise a single bead. The barcoded oligonucleotidesare released from the bead, and the poly-T segment of the barcodedoligonucleotide hybridizes to the poly-A tail of the RNA as in operation1050. The poly-T segment is extended in a reverse transcription reactionas in operation 1052. The thermal cycling conditions are one cycle at42° C. for 2 hours and one cycle at 70° C. for 10 min. Following thermalcycling, the emulsion is broken and the remaining operations areperformed in bulk. A second strand is then synthesized by primerextension as in operation 1054. The reaction conditions for this primerextension include cDNA as template and extension primer. The thermalcycling conditions are one cycle at 98° C. for 3 min and one cycle of98° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 30 min. Followingthis primer extension, the second strand is purified with 0.6×SPRI. Asin operation 1056, in vitro transcription is then performed to produceRNA transcripts. In vitro transcription is performed overnight, and thetranscripts are purified with 0.6×SPRI. The RNA yields from in vitrotranscription are shown in FIG. 16.

Example V: Delivering Lysis Agent to a Partition Using Gel Beads

A lysis agent is introduced into the partition (GEM) via the gel beadsuspension (GBS). The lysis agent is a surfactant that causes wettingfailures (uncontrolled droplet formation) to occur when itsconcentration in the GBS exceeds a threshold.

A larger gel bead can be used to increase the in-partition concentrationof the lysis agent, without increasing the in-GB S concentration (toavoid wetting failures) and without decreasing the total volume of thepartition (which may not be reduced without decreasing the sensitivityof the assay) (FIG. 36A). Alternatively, a larger gel bead can be usedto increase the volume of the partition (which increases the sensitivityof the assay) and preserve the existing in-partition lysis agentconcentration without increasing the in-GB S concentration.

The size of the gel bead can also affect how cells are partitioned. Byreplacing a portion of the sample volume (Z2) with the gel beadsuspension volume (Z1), larger gel beads decrease the in-partitionconcentration of cells, which, according to Poisson statistics, resultsin a lower probability of the unfavorable encapsulation of more than onecell per partition (FIG. 36B).

Example VI: Producing CD3 Protein Conjugated with Short ssDNA Molecules

The CD3 protein and the ssDNA molecule are first activated for clickchemistry reaction. The CD3 protein is activated with5-(methacrylamido)tetrazole (MTet) and the ssDNA molecule is activatedwith trans-cyclooctene (TCO). The ssDNA molecule comprises a biotingroup. The activated CD3 protein and ssDNA molecule are mixed forconjugation by click chemistry reactions. The ssDNA moleculeconcentration is 5 times excess over the CD3 protein concentration toavoid multiple barcode copies conjugating on the same protein molecule.In some cases, the ssDNA concentration is 10 times excess over the CD3protein to maximize barcode attachment. A biotin group may also beincorporated in the activated CD3-ssDNA conjugate for purification. TheCD3 protein and ssDNA conjugate is purified and tested as shown in FIG.37.

Example VII: Labelling Jurkat Cells with Human CD3 and Mouse CD3

The impact of DNA conjugation on the binding of CD3 on Jurkat cells istested. Human CD3 (hCD3, MCA463) and mouse CD3 (mCD3, MCA500) areincubated with AF488-NHS, where the concentration of AF499-NHS is 1×,2×, 5×, and 10× excess over the CD3 protein, in order to generatelabeled CD3, where the AF999 is coupled to an amine of the CD3. Theconjugated hCD3 and mCD3 are incubated with Jurkat cells. Unbound CD3proteins are washed away. The fluorescence signals from the labeledcells are determined (FIG. 38). The fluorescent signals are normalizedby comparing to commercial Jurkat cells control. The data show thatJurkat cells specifically bind to hCD3 over mCD3, indicating that theconjugation of dye/DNA does not affect the binding of CD3 proteins withJurkat cells. Blocking reagents (e.g., FBS, 5% BSA) may be added toimprove specificity.

Example VIII: Conjugating a DNA Barcode to IgG of an Antibody

An antibody is incubated with Methyltetrazine-PEG5-NHS Ester at roomtemperature for 1 hour and desalted. A DNA barcode of about 65 nt longis incubated with TCO-PEG4-NHS Ester at room temperature for an hour anddesalted. The resulting antibody and DNA barcode are incubated at roomtemperature for 2 hours for conjugation. FIG. 39A shows the conjugationstrategy. The conjugated antibody-DNA complex is subject to protein gelanalysis. As shown in FIG. 39B, protein gel shifts of about 20 kDaindicates successful conjugation of the DNA barcode to IgG of theantibody. Multiple viable chemistries for primary antibody barcoding arevalidated (e.g., mTet, dibenzocyclooctyne (DBCO), SiteClick). Theconjugated antibody-DNA complex is incubated with cells for labelling.

Example IX: Conjugating Oligonucleotides to Antibodies UsingAntibody-Binding Proteins

Antibody-binding proteins Protein X (Protein A or Protein G) arefunctionalized with dibenzocyclooctyne-N-hydroxysuccinimidyl ester(DBCO-NHS). Fluorescein amidite (FAM)-labeled oligoX22-azide (3eq) isused as the oligonucleotides to be conjugated with the antibody-bindingproteins. The functionalized antibody-binding proteins and theoligonucleotides are conjugated as shown in FIG. 40. The degree ofconjugation between the dibenzocyclooctyne (DBCO) and Protein G may becontrolled based on Gong et al., Simple Method To PrepareOligonucleotide-Conjugated Antibodies and Its Application in MultiplexProtein Detection in Single Cells. Bioconjugate Chem., 2016, which isincorporated herein by reference in its entirety. Degree of DBCOincorporation may be controlled by adjusting input DBCO-NHSconcentration as shown in FIG. 41.

Moreover, the degree of conjugation may be controlled througholigonucleotide equivalence as shown in FIG. 42. A crudeprotein-oligonucleotide conjugation reaction was analyzed by gelelectrophoresis (SDS-PAGE) to determine conjugation efficiency and thenumber of oligonucleotides conjugated. Increase of oligonucleotideequivalence with respect to the protein leads to a higher degree ofconjugation as shown in FIG. 42. Because the oligonucleotide contains afluorescent molecule, the unused oligonucleotide can easily bevisualized with in-gel fluorescence imaging (black panel in FIG. 42).

The oligonucleotide-Protein X conjugates are incubated with CD47antibodies to form labeled antibodies. The labeled antibodies areincubated with Jurkat cells and washed twice to make labeled cells. Thelabelling of cells is measured by fluorescence signals using flowcytometry (FIG. 43).

Example X: Producing a Bead Coupled with Oligonucleotides with DifferentPrimer Sequences

This example shows a method for producing a bead coupled witholigonucleotides with different primer sequences. The work flow is shownin FIG. 44. A barcode sequence 4421 is ligated to a sequence primer R14411 coupled to a bead. The R1 primer 4411 and barcode sequence 4421form the backbone 4420 of the oligonucleotides on the bead. A pluralityof backbone oligonucleotides 4420 are coupled to the same bead.Different primers sequences are then ligated to the backboneoligonucleotides 4420. The primers include a poly-T primer 4431 thattargets the poly-A of mRNA molecules. The primers also include a targetspecific primer, e.g., an antibody target primer that binds to a barcodeon an antibody. After the second ligation, the bead comprisesoligonucleotides with poly-T primers (4430) and oligonucleotides withantibody target primers (4440). The resulting product from the method isa bead coupled with a plurality of oligonucleotides (FIG. 45A). All ofthe oligonucleotides comprise the same backbone. Some of theoligonucleotide comprises poly-T primers and some comprises the antibodytarget primers. Beads with 0%, 5%, 15%, and 25% of coupledoligonucleotides containing antibody target primers are analyzed by gelelectrophoresis (FIG. 45B)

Example XI: Barcoding Antibody Labelling Agents and Cell Surface FeatureAnalysis

In a first set of experiments, a barcoded oligonucleotide comprising anazide functional group and a FAM dye was conjugated to a Protein Glabelling agent using a click chemistry reaction scheme. The barcodedoligonucleotide included a barcode sequence that may be used to identifyProtein G and also a sequence that may be used as a priming site.Protein G was mixed with increasingly higher molar equivalents ofDBCO-NHS (OX, 1×, 2×, 4× and 6×) in a series of mixtures. The DBCO-NHSwas used to activate amine groups to become reactive to azide. Alsoincluded were varying equivalents of azide oligonucleotide to DBCO (OX,1×, 1.5× and 2×) in the mixtures. Reactions were then allowed to proceedfor 4 hours and the reaction mixtures evaluated with gel electrophoresison a 4-12% bis-Tris gel. The results of the analysis are graphicallydepicted in FIG. 47. Protein G having up to 6 oligonucleotides linkedwere observed.

The various labeled Protein G moieties were then mixed with CD47antibody to bind the labeled Protein G moieties to CD47 antibodies. Theresulting Protein G-CD47 complexes were then incubated with 293T cellssuch that the complexes may bind CD47 on the surface of cells. Cellswere washed to remove unbound complex and then subject to flow cytometryto observe binding of antibodies via the oligo-bound FAM dye. Results offlow cytometry are graphically depicted in FIG. 48.

Next, labeled cells were mixed with a bead coupled to an oligonucleotidecomprising a nucleic acid barcode sequence, a UMI and a poly-T sequencecapable of binding the poly-A sequence of mRNA transcripts in a cell.Also included was a barcoded primer having a priming sequence capable ofspecifically hybridizing the barcoded oligonucleotide coupled to CD47antibodies via the barcoded oligonucleotide's priming site. The mixturewas then partitioned into a droplets in an emulsion. The emulsion wasthen subject to conditions suitable for priming sequences to hybridizewith their respective targets (mRNA or barcoded antibodyoligonucleotide) and for extension of primers via the action of apolymerase or reverse transcriptase. Extension generated barcodedconstructs. Following reactions, the emulsion was broken. Barcodedtranscript constructs still attached to beads were removed by removingbeads and the supernatant subject to 2X SPRI separation to recover the˜110 bp antibody barcode. The recovered products were then analyzed,with results shown in FIGS. 49A and 49B.

Example XII: Coupling of Barcodes

In a bulk experiment, two oligonucleotides shown in FIG. 51A, 5101 and5102, were linked together via extension reactions. Oligonucleotide 5101represented an oligonucleotide comprising a barcode sequence that may beused to identify a partition comprising the oligonucleotide 5101 andoligonucleotide 5102 represented an oligonucleotide comprising a barcodesequence that may be used to identify a labelling agent, such as anantibody coupled to oligonucleotide 5102. Oligonucleotide 5102 alsoincluded a FAM dye and a 3′ reverse complement of a template switcholigonucleotide spacer-rGrGrG region included on oligonucleotide 5101.In the experiment, 50 nM AbBC of oligonucleotide 5102 was mixed witholigonucleotide 5101 in two separate mixtures. Included in the mixturewere reagents for conducting a primer extension reaction, including oneof two reverse transcriptases capable of facilitating a primer extensionreaction and dNTPs. Extension products were then analyzed via capillaryelectrophoresis.

The results of the experiment are graphically shown in FIG. 51B. Asshown, expected extension products having both a sequence correspondingto the barcode sequence of oligonucleotide 5101 (or a complement of thebarcode sequence) and a sequence corresponding to the barcode sequenceof oligonucleotide 5102 (or a complement of the barcode sequence) weredetected. These results confirm that the reverse transcriptases testedmay be used to generate extension products having sequencescorresponding to both barcode sequences of oligonucleotides 5101 and5102.

Example XIII: Single-Cell Barcode Behavior

Anti-CD47 and Anti-CD99 antibodies were obtained and both types werecoupled to an oligonucleotide comprising a barcode sequence that wassuitable for identifying its respective antibody and also comprising aunique molecular identification (UMI) sequence and a template switcholigonucleotide reverse complement sequence (e.g., C C C). Theantibody-oligonucleotide constructs were generated by linking theoligonucleotides to protein G and then binding the proteinG-oligonucleotide constructs to the antibodies. The oligonucleotideswere linked to protein G by modifying protein G with a single cysteineresidue and linking it to oligonucleotides via the cysteine residue.Protein G also included a Hisx6 tag which may be used to separateunconjugated oligonucleotides from those coupled to Protein G. Sampledata from gel electrophoresis analysis of generated constructs is shownin FIG. 52A. The lanes in FIG. 52A show expression of acysteine-containing protein G antibody binding protein. The culture lanedepicts a homogenized cell culture, the flow through lane depicts is allproteins that did not bind to a nickel-NTA column, and the two elutionlanes are eluted purified protein G.

Jurkat cells were then incubated with antibody-oligonucleotideconstructions to bind antibodies to the surface of cells via theirrespective cell surface feature targets. The cells were then partitionedinto aqueous droplets in an emulsion, along with beads linked tooligonucleotides comprising a barcode sequence, a UMI sequence, apriming sequences capable of hybridizing with antibody-boundoligonucleotides (e.g., primer sequence include a template switchsequence, such as rGrGrG). A reducing agent, capable of disruptingdisulfide linkages of beads and linkages between beads and itsoligonucleotides was also included in the partitions. The reducing agentreleased the bead's oligonucleotides and the droplets were thensubjected to conditions suitable for hybridizing the previouslybead-bound oligonucleotides to cell-bound antibody oligonucleotides viaan interaction of sequences of the two oligonucleotides, including viaan rGrGrG/CCC interaction. While a particular sequence is shown,hybridization may be achieved via any constant sequence at the ends ofthe two oligonucleotides.

The two hybridized oligonucleotides were then extended in primerextension reactions to generate constructs comprising sequencescorresponding to both bead oligonucleotide and antibody barcodesequences, similar to the example scheme shown in FIG. 52B (panel I).The emulsion was then broken, the extended products further processedand then subject to sequencing. Sequencing results for Jurkat+CD47 andJurkat+CD47/CD99 runs are graphically depicted in panels I and II,respectively, of FIG. 53A and tabulated in FIG. 53B. The data shown inFIG. 53A and FIG. 53B indicate that the antibody-oligonucleotideconstructions comprising barcode sequences were able to show single cellbehavior, as evidenced, for example, by an approximately 2-logenrichment of antibody-oligonucleotide UMIs in bead-originating barcodeconstructs corresponding to cells.

Example XIV: Antibody Barcode Staining Parameters

Various parameters associated with methods described herein wereevaluated in the context of their effects on antibody-barcode constructbinding, including a reverse transcription deactivation process and theconcentration of reducing agent in partitions (e.g., reducing agent usedto degrade barcoded beads as described elsewhere herein).

Reverse transcription can be deactivated by elevating the temperature ofreverse transcription reaction mixtures to relatively high temperatures(a “heat kill”). However, such high temperatures may result inantibody-barcode constructs precipitating out of reaction mixtures,resulting in an inability to bind to cells. Various anti-CD3 barcodeconstruct samples were tested against cells, with some samples subjectto heat kill and others not subjected to heat kill. Sequencing data forthe experiments is tabulated in FIG. 54. As shown in FIG. 54, a numberof sequencing metrics are improved when no heat kill is used, includingreads mapped confidently and complexity.

Moreover, high concentrations of reducing agents can also degradeantibodies used to label cell-surface features. Accordingly, the effectof lower reducing agent (e.g., DTT) concentration by 10-fold was testedon overall efficiency of reverse transcription in partitions. As show inFIG. 55, traces are similar for all samples tested (22 mM DTT vs. 2.2 mMDTT), suggesting that reverse transcription, as described elsewhereherein, can effectively proceed at substantially reduced DTTconcentrations. In another experiment, 0.15 mM DTT was also shown to beeffective.

While some embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

1.-30. (canceled)
 31. A method for multi-analyte processing or analysis, comprising: (a) providing a mixture comprising a first analyte, a second analyte that is different than said first analyte, and a support having coupled thereto (i) a first barcode molecule capable of coupling to said first analyte and (ii) a second barcode molecule capable of coupling to said second analyte; (b) in said mixture, (i) using said first analyte and said first barcode molecule to synthesize a first barcoded product, and (ii) using said second analyte and said second barcode molecule to synthesize a second barcoded product; and (c) characterizing (i) said first barcoded product or derivative thereof and (ii) said second barcoded product or derivative thereof, to identify said first analyte and/or said second analyte.
 32. The method of claim 31, wherein said support is a bead.
 33. The method of claim 32, wherein said bead is a gel bead.
 34. The method of claim 31, wherein, after (a), said first barcode molecule and/or said second barcode molecule is released from said support.
 35. The method of claim 31, wherein said first analyte and said second analyte are differently selected from the group consisting of a deoxyribonucleic acid molecule, a ribonucleic acid molecule, and a labelling agent capable of coupling to a protein.
 36. The method of claim 35, wherein said protein is a surface feature of a cell.
 37. The method of claim 35, wherein said labelling agent further comprises a labelling agent barcode molecule, wherein said first barcode molecule or said second barcode molecule is capable of coupling to said labelling agent barcode molecule.
 38. The method of claim 37, wherein said labelling agent barcode molecule is used to synthesize said first barcoded product or said second barcoded product.
 39. The method of claim 37, wherein said labelling agent is an antibody.
 40. The method of claim 31, further comprising, prior to (c), removing (i) said first barcoded product or derivative thereof and (ii) said second barcoded product or derivative thereof from said mixture.
 41. The method of claim 31, wherein said first barcode molecule is a first nucleic acid molecule comprising a first nucleic acid barcode sequence, and wherein said second barcode molecule is a second nucleic acid molecule comprising a second nucleic acid barcode sequence.
 42. The method of claim 41, wherein said first nucleic acid barcode sequence comprises a first barcode sequence segment, and wherein said second nucleic acid barcode sequence comprises a second barcode sequence segment that is identical to said first barcode sequence segment.
 43. The method of claim 41, wherein said first barcoded product and said second barcoded product are nucleic acid molecules, wherein said first barcoded product comprises said first barcode sequence, and wherein said second barcoded product comprises said second barcode sequence.
 44. The method of claim 43, wherein said characterizing comprises sequencing (i) said first barcoded product or a derivative thereof and (ii) said second barcoded product or a derivative thereof, to identify said first barcode sequence and said second barcode sequence.
 45. The method of claim 41, wherein said first or said second analyte is a labelling agent capable of coupling to a protein, and wherein said labelling agent comprises a labelling agent nucleic acid molecule coupled thereto.
 46. The method of claim 45, wherein said labelling agent nucleic acid molecule coupled to said labeling agent comprises a peptide barcode sequence, and wherein said peptide barcode sequence identifies said protein.
 47. The method of claim 41, wherein said first barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte, and wherein said second barcode molecule comprises a second priming sequence capable of hybridizing to said second analyte.
 48. The method of claim 47, wherein said first priming sequence or said second priming sequence is an oligo(dT) sequence, a random N-mer sequence, or a sequence complementary to a sequence in a labelling agent nucleic acid molecule coupled to a labelling agent, which labelling agent is capable of coupling to a protein in said mixture.
 49. The method of claim 31, wherein said mixture is in a partition among a plurality of partitions.
 50. The method of claim 49, wherein said partition is a droplet and said plurality of partitions is a plurality of droplets.
 51. The method of claim 49, wherein said partition is a well and said plurality of partitions is a plurality of wells.
 52. The method of claim 49, wherein said partition comprises said first analyte, said second analyte, and said support.
 53. The method of claim 52, wherein said partition further comprises a cell, wherein said first analyte and/or said second analyte are associated with said cell.
 54. The method of claim 52, wherein said first barcode molecule is a nucleic acid molecule comprising a first nucleic acid barcode sequence, wherein said second barcode molecule is a nucleic acid molecule comprising a second nucleic acid barcode sequence, and wherein said first barcode molecule and said second barcode molecule are releasably attached to said support.
 55. The method of claim 54, wherein said support is a bead.
 56. The method of claim 55, wherein said bead is a gel bead.
 57. The method of claim 54, wherein after (a), said first barcode molecule and said second barcode molecule are released from said support.
 58. The method of claim 57, wherein said first barcode molecule and said second barcode molecule are released upon application of a chemical stimulus.
 59. The method of claim 58, wherein said chemical stimulus is a reducing agent.
 60. The method of claim 58, wherein said partition further comprises said chemical stimulus. 